Microfabricated recessed disk microelectrodes: characterization in static and convective solutions

ABSTRACT

Construction and characterization of microfabricated recessed disk microelectrodes (RDMs) of 14 and 55 μm diameter are reported. The work reported here makes several new contributions to the current literature on microfabricated RDMs. Hybrid blamers were constructed by fusion of vesicles of dimyristoylphosphatidyl choline (DMPC), which forms the top layer, with ethanol-rinsed SAMs of hexadecanethiol on gold, which form the bottom layer. Gramicidin A was included in the modifying solutions to incorporate it into hybrid blamers.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/946,259, filed on Sep. 4, 2001, which is acontinuation-in-part of U.S. patent application Ser. No. 09/775,937,filed Feb. 2, 2001, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/255,950, filed Feb. 23, 1999, which claimspriority to U.S. provisional application Serial No. 60/075,955, filedFeb. 23, 1998. This application claims priority to U.S. provisionalapplication Serial No. 60/055,527, filed Aug. 8, 1997. This applicationis also a continuation to U.S. patent application Ser. No. 09/071,356,filed Apr. 30, 1998, which claims priority to U.S. provisionalapplication Serial No. 60/042,100, filed Apr. 30, 1997. Each of theseapplications are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

REFERENCE TO A SEQUENCE LISTING, A TABLE OR A COMPUTER PROGRAM LISTINGCOMPACT DISC

[0003] Not applicable.

BACKGROUND OF THE INVENTION

[0004] 1. Field of the Invention

[0005] The present invention relates to microfabricated recessed diskmicroelectrodes. More specifically, this invention relates tomicrocavities containing microelectrodes and being separated fromsurrounding media by a lipid bilayer that is anchored to the rim of themicrocavity. The invention also relates to microcavities having a holein the bottom in order to relieve osmotic pressure. The invention alsorelates to arrays of such microcavities.

[0006] 2. Prior Art

[0007] Microelectrode based electrochemical analysis systems areadvantageous over systems containing macroelectrodes. First,microelectrodes can be made very small, for example bands with widths 32nm and single microdisks with diameters of 2 nm. Second, the currentdensity for microelectrodes is greater than that at macroelectrodes dueto radial diffusion. This results in a measurable steady state currentat electrodes of the dimensions described above. Finally, uncompensatedresistance does not induce large potential drops due to the smallcurrents drawn by microelectrodes.

[0008] Microelectrodes have been used for analysis in small volumes. Twogeneral approaches have been used for the analysis, differentiated bythe construction of the system. The first type of system uses singleelectrodes placed in solution. The second type of system usesmicrofabrication techniques to pattern the electrodes. The smallestvolumes, 0.6 nL, using this technique have been demonstrated.

[0009] Microelectrodes have been used for analysis in convectivesystems. One example of this is the use of microelectrodes as detectorsin liquid chromatography and capillary electrophoresis. The baselinenoise, which also helps determine the limit of detection is dependentupon the flow rate (i.e. convection). A number of different geometrieshave been reported, with the band and disks being among the most common.

[0010] Microelectrodes, in general, have been studied extensively andreviews have been published. Microelectrodes have been used in complexmedia such as blood and urine. They have also been used to providespatially-resolved information from surfaces and cell membranes. A hostof biosensor applications has been reported.

[0011] The most commonly studied microelectrode geometry is the diskbecause it is relatively simple to construct and can attain true steadystate current. Both planar (PDM) and recessed disk (RDM) microelectrodeshave been studied. A recessed microdisk resides at the bottom of acavity whose walls are made of insulator material. Although the currentmeasured at RDMs is typically less than that at PDMs of equal radius, itcan be independent of convection outside of the cavity, depending uponthe cavity's dimensions and the strength of the convective forces. Twocomponents give an RDM its unique properties: the size of the electrodeand its position, which is set back from the main plane of theinsulating layer of the substrate.

[0012] Recessed microdisk electrodes were originally constructed fromin-plane microdisk electrodes. Either chemical or electrochemicaletching has been used to etch the electrodes away from the surface planeof the insulator. The depth of the cavity and surface roughness of theseelectrodes are difficult to control. The early applications includedchemical measurements in convective systems.

[0013] The incorporation of membrane proteins and enzymes into modifyinglayers on surfaces is of interest for model systems of biomembranes andfor the development of chemical sensors. Membrane protein structure andfunction are highly dependent on the surrounding environment, and thus,it is essential to design materials on surfaces that provide thenecessary characteristics to host such proteins. Langmuir-Blodgett (LB)techniques have been used to assemble phospholipids onto surfaces toprovide biomembrane-like environments. These assemblies have beencharacterized by AC impedance measurements, X-ray photoelectronspectroscopy (XPS), and atomic force microscopy. Another more recentmethod that has the advantage over LB methods of ease of formationinvolves vesicle fusion to surfaces to form hybrid blamers. In thismethod, the chemistry of the strong interaction of sulfur with gold isused to form self-assembled monolayers (SAMs) to produce a first layer.This layer provides the driving force for deposition of a second layerof phospholipids by hydrophobic coupling of phospholipid vesicles to theSAM layer. The first layer is chemisorbed rather than physisorbed andhighly ordered, which provides a more ordered foundation for additionalmultilayer construction. Also in forming the second layer, a waterrinse, rather than an organic solvent rinse, flushes away unboundvesicles and lipids without removing biomolecules incorporated withinthe surface-confined layers.

[0014] An area of great importance is the incorporation of biologicalmolecules onto electrodes to aid in electrochemical detection ofanalytes. An important aspect is the chemistry behind modifying theelectrode without destroying the activity of the biological molecule,many chemical methods have been developed to modify electrode surfaces.Immobilizing methods for biological molecules such as enzymes includecovalent bonding, adsorption, monolayer deposition, entrapment, andmicroencapsulation. Currently, SAMs are being studied as immobilizationtools because of the ease with which they create highly ordered organicfilms. A recent example is use of SAMs of alcohol-terminaledalkanethiols and glucose oxidase on gold electrodes to prepare a glucosesensor.

[0015] Providing the necessary hydrophobic and hydrophilic properties onthe electrode surface is a challenging problem. The native environmentfor many proteins and enzymes is the cellular biomembrane. Severalmethods are being developed to artificially recreate that environment onelectrode surfaces. Surface-confined lipid membranes on electrodes havebeen formed with Langmuir-Blodgett (LB) techniques and by combining SAMswith phospholipid vesicles. Controlling access to the underlying surfacehas been demonstrated using gate sites through a monomolecular LB film.

[0016] Bilayer formation using SAMs and phospholipid vesicles has beenstudied by electrochemistry and surface plasmon resonance. properties ofhybrid blamers by cyclic voltammetry using Fe(CN)₆ ³⁻ as the redoxspecies in an electrolyte solution of 1 M KCl and determined that thepresence of the hybrid bilayer reduces the rate of electron transfer byapproximately two orders of magnitude from that of the bare electrode.Plant has also compared capacitance values obtained by impedancemeasurements of SAMs of alkanethiols to those of hybrid blamers ofoctadecanethiol (C₁₈SH) and 1-palmitoyl-2-oleoylphosphhatidylcholine(POPC) and reports that hybrid blamers are sufficiently flexible toaccommodate a molecule such as a pore forming mellitin. A glucose sensorthat is based on a similar bilayer-self assembling technique has alsobeen reported. This method involves using tetracyanoquinodimethane(TCNQ) that resides within a dodecanethiolphosphatidylcholine andphosphatidylethanolamine bilayer, and serves as a mediator between theunderlying electrode and overlying, cross-linked glucose oxidase.However, the bilayer thickness, estimated from impedance measurements,was smaller than typical values reported in the literature, and the TCNQdiffused out of the bilayer during electrochemical measurements.

[0017] Membrane assembly methods have been used in conjunction withenzyme reconstitution procedures to modify the electrode surface andstudy the electron-transfer reaction of immobilized bovine cytochrome coxidase. Cyclic voltammetry and potential step chronoabsorptometry wereused to show the direct electron transfer between the gold substratesand the cytochrome c oxidase incorporated in a dodecanethiol and1-palmitoyl-2-oleoylphosphhatidylethanolamine (POPE) and POPC layers. Inaddition, the immobilized enzyme was shown to both reduce and oxidizethe cytochrome c in solution. Others have used vesicles formed frommolecules with head groups having a net positive or negative charge suchas dimethyldioctadecylammoniumbromide (DODAB) and dimyristoylphosphatidylglycerol (DMPG), respectively. Lipid blamers and trilayerson solid supports can be formed by fusing these charged vesicles to acharged monolayer such as carboxylate mercaptans directly or via acation linkage. These layers have been analyzed by impedance and surfaceplasmon resonance to determine the mean thickness of the membranes.Impedance analysis combined with spectroscopy has provideddiscrimination in identifying between specific and non-specificadsorption of streptavidin and biotinated-lipids.

[0018] Techniques to create supported blamers using vesicles that form atop fluid layer of phospholipids onto a fixed SAM have also beendemonstrated. Permeation of ions through these blamers was studied uponincorporating the pore-forming peptide mellitin. A glucose sensor thatis based on a similar bilayer-self assembling technique has also beenreported. The assembly involved tetracyanoquinodimethane which resideswithin an alkanethiol/phospholipid bilayer, and serves as a mediatorbetween the underlying electrode and overlying, cross-linked glucoseoxidase.

[0019] Because of its simple composition and characteristic functiondependence on structure, Gramicidin A is used as a convenient probe toevaluate modifying layers on electrodes. This small ion channel-formingpeptide is one of the best characterized and most extensively studiedmembrane polypeptides. It is an antibiotic that is isolated fromBacillus brevis and is active against Gram-positive bacteria. Itconsists of an alternating L, D pentadecapeptide with the primarysequence ofHCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-(L-Trp-D-Trp)₃-L-Trp-NHCH₂CH₂OH.The 3-dimensional conformation of gA is complex and dependent upon itsenvironment. In biological or model membrane systems, gA adopts an ionchannel conformation which allows the passage of water and small,monovalent cations. The channel is in the form of two β-helical monomersthat dimerize end to end with the formyl-NH ends associated in thecenter of a lipid bilayer. The length of the dimer is approximately 26Å. The peptide backbone forms a hydrophilic pore that has a diameter ofabout 4 Å. Gramicidin A has been characterized in only a fewelectrode-modified systems. Evidence has been presented of theselectivity of gA toward metal mono cations on electrode surfaces.Gramicidin was incorporated into dioleoyl phosphatidylcholine and bovinebrain phosphatidylserine monolayers using LB techniques on mercury dropelectrodes. By cyclic voltammetry, selective permeability to TI⁺ overCd²⁺ for layers containing gA is consistent with the gA being in the ionchannel conformation. This system is not easily conducive to furtherevaluations by spectroscopy due to the nature of mercury. Differentpreparation techniques for supported lipid layers have been evaluated byimpedance analysis. Gramicidin has been incorporated into one type offilm, a SAM of 3-mercaptopriopionic acid, covered with a bilayer ofDODAB, formed from fusion of DODAB vesicles, to create trilayer films.Again, spectroscopic characterization was not performed. However,electrochemical behavior was observed in the presence of Cs⁺ and Sr²⁺that might be interpreted as gramicidin channels controlling ionpermeation through the film.

[0020] It is therefore desirable to produce both tubular nanoband andrecessed disk microelectrodes within a microcavity capable of detectingelectrical currents undistorted by convection of a solution.

[0021] It is also desirable to produce microcavities havingmicroelectrodes and a lipid bilayer extending across the top of themicrocavity.

[0022] It is also desirable to produce microcavities havingmicroelectrodes, a lipid bilayer, and a hole in the bottom to reduceosmotic effects.

[0023] It is also desirable to produce arrays of microcavities.

[0024] It is also desirable to develop an accurate, efficient andreproducible method for creating microcavities or arrays thereof havingmicroelectrodes, lipid blamers and holes to reduce osmotic effects.

BRIEF SUMMARY OF THE INVENTION

[0025] Construction and characterization of microfabricated recesseddisk microelectrodes (RDMS) of 14 and 55 μm diameter are reported. Forevaluation of electrode function, both faradaic current in Ru(NH₃)₆ ³⁺solution and charging current in KNO₃ solution were measured with cyclicvoltammetry. The experimental maximum current was measured and comparedto values calculated, assuming radial and linear diffusion. At slow scanrates (0.1 Vs⁻¹), where radial diffusion dominates, the steady statemeasured with 55 μm RDM is 53.5±0.48 nA and the 14 μm RDM is 5.39±0.96nA. The calculated current based on recessed disk theory for thesediameters of electrodes is 34.9 and 6.10 nA respectively. The calculatedcurrent based upon planar recessed disk microelectrode theory is 41.4and 10.5 nA respectively. At fast scan rates (204 Vs⁻¹), where lineardiffusion dominates, the current measured with the 55 μm RDM is784.6±42.0 nA and the 14 μm RDM is 35.4±9.5 nA. The predicted current,based on linear diffusion, is 1274 nA and 82.6 nA, respectively. Thelarge deviations at fast scan rates are due to uncompensated resistance.The dependence of capacitance on scan rate of the RDMs was found to besimilar to that of a macroelectrode, indicating good adhesion betweenthe insulator and the electrode. Also, the application of the RDMs toconvective systems is discussed. Chronoamperometry of Ru(NH₃)₆ ³⁺ inKNO₃ in both static and stirred solutions was performed using the RDMsand the current is compared to those from a planar disk microelectrode(PDM). The signal-to-noise ratio of the RDM compared to the PDM is onaverage four times greater for both of the stirred solutions.

[0026] The work reported here makes several new contributions to thecurrent literature on microfabricated RDMs. First, the microfabricatedRDMs are smaller (14 and 55 μm diameters) than those reported by others(˜1 mm), with greater depth-to diameter ratios (0.29 and 0.07, comparedto 0.015 and 0.04), which should improve performance in convectivesystems. Scanning electron microscopy was used to evaluate the generalshape and quality of the cavities. Second, we present a detailedevaluation of the elctrochemical responses of the mircofabricated RDMsand compare them to theory. Although characterization has been performedpreviously on RDMs that were not microfabricated, conclusions based onthose studies may not be valid for microfabricated RDMs. A differentbehavior may result because of the difference in materials used andextensive processing involved. The electrochemical response in Ru(NH₃)₆³⁺ is compared to theory for linear and radial diffusion. Capacitancewas determined from cyclic voltammetry in 0.5 M KNO₃ and is compared tothat for a macroelectrode. This latter comparison elucidates the qualityof the seal between the insulator and the electrode. Third, evidencefrom chronoamperametry in stirred Ru(NH₃)₆ ³⁺ solution shows theadvantages of microfabricated RDMs vs. PDMs in convective systems.

[0027] The characterization and application of a cavity electrode system(CES) containing individually-addressable recessed microdisk and tubularnanoband electrodes is discussed. Two diameters of CES, 13 and 53 μm,are described. The depth of each cavity is 8 μm. Each of the electrodesis characterized in Ru(NH₃)₆ ³⁺ and KNO₃ solution at 0.1 Vs⁻¹. Theexperimental current measured at the electrodes in the 53 μm CES werewithin error of models for radial diffusion to the respective geometry.The experimental current for both electrodes in the 13 mm CES deviatedfrom the models. The band electrode exceeded the model (6.31±0.28 nAcompared to 3.98 nA). The disk electrode was less than the modelpredicted (2.13±0.46 nA compared to 3.81 nA). The formation andstability of a Ag/AgI pseudo reference electrode on the band electrodeis shown. The E^(o) for Ru(NH₃)₆ ^(3+/2+) measured with the Ag/AgIelectrode is −0.053±0.016 V. The reference electrode was found to bestable over multiple experiments without supporting electrolyte. Thecomplete electrochemical cell (working electrode, reference andauxiliary electrode) was used for analysis in small volumes (1 and 10μL) of hydroquinone and Ru(NH₃)₆ ³⁺. Finally, the CES was used instirred solutions. The signal-to-noise ratio (SNR) from 13 μm CES showedno dependence upon stir rate up to 150 rotations per minute (rpm). TheSNR from the 55 μm CES showed only a small change with stir rates up to150 rpm.

[0028] Hybrid blamers were constructed by fusion of vesicles ofdimyristoylphosphatidyl choline (DMPC), which forms the top layer, withethanol-rinsed SAMs of hexadecanethiol on gold, which form the bottomlayer. Gramicidin A was included in the modifying solutions toincorporate it into hybrid blamers. Results from Polarization-modulationFourier-transform infrared :o reflection-absorption spectroscopy(pM-FTIRRAS) and X-ray photoelectron spectroscopy (XPS) on such hybridblamers are reported for the first time. A comparison is made betweenthose results and ellipsometric and electrochemical measurements in KNO₃and Mg(NO₃)₂ solutions. Capacitance determinations by cyclic voltammetry(at 0.0 and 0.4 V VS. Ag/AgCl) and AC impedance (at 0.0 V vs. Ag/AgCl)are discussed. PM-FTIRRAS and ellipsometry reproducibly demonstrate thatblamers are indeed formed. XPS analysis of the blamers showed evidenceof the presence of DMPC and gA, although results were not veryreproducible, presumably due to sample damage during analysis. Thecapacitance in KNO₃ solution for the gA-containing bilayer is higherthan that for blamers without gA. The opposite trend occurs forsolutions of Mg(NO₃)₂. PM-FTIRRAS and XPS of the SAM layers alone,assembled in the presence of gA do not show evidence of the presence ofgA. However, those SAMs exhibit higher relative capacitance in KNO₃, buta lower relative capacitance in Mg(NO₃)₂ than SAMs assembled in theabsence of gA.

[0029] Gramicidin A was assembled into organic films on electrodes tocreate and study possible materials for electrochemical sensing Oneassembly method involves self-assembled “monolayers” (SAMs) fromhexadecanethiol (C₁₆SH)+gA mixtures followed by different solventrinses. Ethanol rinses yield monolayers, but appear to remove gA. Waterrinses form multiple layers of C₁₆SH and gA. A second assembly methodreproducibly forms blamers by disruption or gA-containing vesicles ordimyristoyl phosphatidylcholine (DMPC) onto ethanol-rinsed SAMs ofC₁₆SH+gA followed by a water rinse. Ellipsometry verified the number orlayers or molecules in the films on the surfaces. Permeation or Fe(CN)₆³⁻ is essentially negligible at all films. Electrochemical responses toK⁺ and Mg²⁺ at blamers and to Ag⁺ and Pb²⁻ at water-rinsed SAMs isconsistent with the selectivity of the channel former of gA. The merepresence of gA might also cause this selectivity. Exact conformation ofgA in these films has not yet been determined.

[0030] Two ways are demonstrated to assemble membrane proteins ontoelectrode surfaces for the purposes of developing chemical sensors andstudying protein-membrane interactions. Gramicidin A is incorporatedinto these assemblies. The gA serves as a probe of the surroundingmolecular environment and allows selective permeation of monovalentcations and blocks multivalent cations and anions. One means of assemblyis the simultaneous self assembly of gA with hexadecanethiol to formmono- or multilayers onto a gold electrode surface. The spontaneousadsorption of organothiols onto gold to form SAMs has become popular dueto its simplicity, versatility, reproducibility and many possibleapplications. The second assembly structure is a supported hybridbilayer that is formed from a combination of SAMs of hexadecanethiolwith gA and vesicles of phospholipids containing gA. Studies on thesemolecular assemblies involved ellipsometry, capacitance measurements,and permeation of redox molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 shows a diagram of a cross-section view (not to scale) of asingle cavity.

[0032]FIG. 2 shows a fabrication schematic, cross-section view.

[0033]FIG. 3 shows scanning electron micrographs of (a) 55 μm and (b) 14recessed disk microelectrodes that are 4 μm deep the 14 μm RDMmicrograph shows some irregularities along the walls of the opening.

[0034]FIG. 4 shows top down and side view schematics of the cavitymicroelectrode system.

[0035]FIG. 5 shows scanning electron micrograph (SEM) of the cavitymicroelectrode system. This top down view shows only layer 5 and layer 1(Au).

[0036]FIG. 6 shows a schematic of CES showing (a) the top-down view oftwo electrodes of the pattern, (b) an enlargement of the region wherethe cavity is located, and (c) a cross section of the cavity.

[0037]FIG. 7 shows how blamers were constructed by allowing phospholipidvesicles to assemble from an aqueous suspension onto ethanol-rinsed,SAM-modified electrodes.

[0038]FIG. 8 shows a top down view of the electrode array showing thelayers and positioning of the individual electrodes. The electrodescolored blue are in the bottom plane and constitute the disk electrodesshown in FIG. 9. The two insulator layers are represented by thecross-hatched areas. The insulator is polymide for these devices. Thered colored electrodes are the second electrode layer. They are thetubular band electrodes in FIG. 9. The top later is gold. This layer wasnecessary to defined a cavity with straight walls. (See FIG. 9) In thisfigure, it is also defined by the cross-hatched area.

[0039]FIG. 9 shows the location and size of the cavities from the topdown view and a side view of a representative cavity showing thearrangement of layers. The cavities are located in the overlap region ofthe two electrode layers (top schematic). The four possible cavitydiameters are shown. The bottom schematic shows the altering of materialinside the cavity. The black section is the silicon substrate that isused to support the electrode system. The blue and red layers correspondto appropriate electrode layers. The cross hatched sections areinsulator (polymide) layers. The green layer is the protective goldlayer that protects the upper lip of the cavity during processing so theshape and vertical walls of the cavity are maintained.

[0040]FIG. 10 shows two kinds of electrode devices and their generaldimensions that will be to discussed in the talk. The top one is only a2-layer device, where lines have been etched through, exposing linearedge band electrodes along the walls and which we have presented in thepast. The bottom figure is of a cross-section of a 5-layer microcavitydevice, which is relatively new.

[0041]FIG. 11 shows the advantage of stacking alternating layers ofinsulator and conductor metal layers to increase the three dimensionalfunctionality of the devices, while using the same substrate area. Thisidea has been presented on many occasions before. The multilayermicroactivity electrode devices beautifully put this idea into practice.

[0042]FIG. 12 shows scanning Electron Microscopy (SEM) images of (a) atop view of chromium, gold, and silicon nitride on top of glass,patterned to form 15, 4 mm band features flanked by 2, 50 mm featuresand (b) a side view of one of the edges which contains a submicron bandelectrode.

DETAILED DESCRIPTION OF THE INVENTION

[0043] Alkanethiols bind to both gold and fatty acids. These qualitiesare taken advantage of in order to create microcavities having lipidblamers extended across their openings. Various membrane proteins may beinserted into these blamers using the methods described below. Thisfacilitates the study of protein mediated membrane transport, especiallyof molecules that are readily susceptible to electrochemical detection.A hole may be made in the bottom of the cavities in order to relieveosmostic pressure, thereby increasing the accuracy of electrochemicaltransport analysis. It is also possible to create large arrays of thesebilayer covered cavities. This allows for faster gathering of more dataand also provides the ability to study a variety of electrochemicallysusceptible molecular species within a single solution sample. Themethods for forming these structures, as well as the physicalcharacteristics of these microcavities is discussed in detail in thefollowing examples:

EXAMPLE 1

[0044] Materials

[0045] All chemicals were reagent grade and used as received. Aqueoussolutions were prepared with high purity deonized water (Mille-Q, modelRG). A gold coin (Credit Suisse, 99.99%) and chromium plated tungstenrod (R. D. Mathis) served as sources for thermal evaporation. Siliconwafers (5″, (100) were obtained from Silicon Quest International (SantaClara, Calif.). Potassium nitrate, sulfuric acid, hydrochloric acid,nitric acid and 30% hydrogen peroxide were purchased from FisherScientific. Hexaamine-ruthenium (III) chloride was obtained fromAlderich Chemical Co. Positive photoresist (AZ330RS) and photoresistdeveloper (AZ00K) were purchased from Hoechst-Celanese. Polymide(Pyralin PI-2721, DuPont) was purchased from DuPont. A gold 10 μmdiameter PDM (BioAnalytical Systems) BAS was used as the control.

[0046] Electrochemical Measurements

[0047] A BAS-100B potentiostat and PA-1 preamplifier controlled withBAS-100W electrochemical software were used to perform cyclicvoltammetry (CV) and chronoamperometry (CA). The electromechemical cellcontained a Pt flag auxiliary electrode and Ag/AgCl (saturated KCl)reference electrode. For CV experiments, a solution of 5.0 mM Ru(NH₃)₆³⁺ and 0.5 M KNO₃ was purged with Ar to minimize the oxygen content.Stirring studies involved CA and were performed on a Corning PC-320 stirplate with ½″ magnetic stir bar (Fisher Scientific). The cell volume was40 mL and was not purged prior to CA. The rotation rate was determinedby counting the rotations of the stir bar over a given period. Aumacroelectrodes were made by depositing 15 Å of Cr followed by 2000 Å ofAu on an oxidized Si wafer. the preparation of the Si wafer andformation of a passivating oxide layer is the same as described belowfor the mircofabrication. Uncompensated resistance measurements weremade using the BAS-100B. A 25 mV DC potential was applied in a 0.5 MKNO₃ solution.

[0048] Construction of Recessed Disk Microelectrodes

[0049] The fabrication or RDMs was accomplished by forming a holethrough a Au and polymide layer exposing an underlying Au Disk. FIG. 1shows a cross-section view of an RDM 100. Bottom Au layer 104 is arecessed disk microelectrode. It is separated from top Au layer 102 byinsulating layer 106. Substrate 108 is typically a silicon wafer, butmay be comprised of a number of other materials. The top later of Au102, while not used in the electrochemical measurements is essential inthe fabrication process so that cavities with well-defined, verticalwalls can be produced. Cavity devices, that accommodate twoindividually-addressable electrodes, are described elsewhere. Here wefocus on the use of the same design for a more simplified electrodeconfiguration.

[0050] The fabrication of RDMs consists of four steps. The process isshown as a cross-section schematic in FIG. 2. A 2 μm SiO₂ film was grownon a Si wafer 114 by thermal oxidation. The wafer was spin-coated with apositive photoresist 112 and exposed to UV light (400 W, 300 nm) througha photolithographic mask (HTA Photomask). The photoresist was developed,leaving the pattern of a series of parallel lines, which eventuallybecome the contact leads and microdisk electrodes. A 15 Å Cr film, whichserves as an adhesion layer, and 1000 Å Au layer 116 were deposited onthe photoresist by thermal evaporation (Edwards 306 Auto). The wafer wassonicated for 15 min in acetone, which dissolves the photoresist,causing lift-off of the metal on top.

[0051] After drying for 30 minutes at 125° C., the wafer was spin-coatedwith polymide 118 (4 μm thick). The polymide was polymerized by exposureto UV light and then cured at 150° C. for 30 minutes and 250° C. for 30minutes to cross-link the polymer. Cr (15 Å) and Au (1000 Å) 120 weredeposited on top of the polymide 118 by thermal evaporation. The waferwas spin-coated with positive photoresist. The photoresist was patternedby UV-exposure through a second photolithographic mask (HTA Photomask).The Au and Cr were etched simultaneously with 50% aqua regia (1 HNO₃:3HCI). The remaining photoresist was stripped with acetone and the waferwas dried for 30 minutes at 125° C. This left a layer of Au/Cr 116covering the electrode lines with an area over the end of the lines leftopen for contact purposes.

[0052] The wafer was spin-coated with photoresist and patterned using athird photolithographic mask (Photronics). This step leaves a circularopening through the photoresist over each region defined by the lines inthe first gold layer. The topmost layer of Au was etched using radiofrequency (RF) sputtering (5 minutes, 50 sccm Ar, 30 mT, 500 V). Thepolymide was etched with reactive ion etching (RIE) (13 min, 40 sccm O₂,10 sccm SF_(6,) 300 mT, 300 W). Before use, the electrodes were cleanedby sonicating in acetone for 30 s. The electrode pattern design includes4 lines of Au underlying the polyimide. Each has one cavity of adifferent nominal diameter: 50 μm, 10 μm, 5 μm, and 2 μm. Only the 50and 100 μm cavities could be formed with this set of microfabricationconditions. Scanning electron microscopy (SEM) was performed with aHitachi S-2300 scanning electron microscope (20 kV acceleratingvoltage). A profilometer (Dektak 3030) was used to measure the polymidethickness.

[0053] Physical Characterization

[0054] Recessed disk microelectrodes were characterized by SEM todetermine shape and dimensions. FIGS. 3a and 3 b show top down electronmicrographs of RDMs of 14 μm 124 and to 55 μm 126 diameter,respectively. The circle defines the edge of the disk at the bottom ofthe cavity. The larger cavity appears to have a smooth, circular openingat 1000× magnification. The opening of the 14 μm diameter cavity seemsless regular (3000× magnification). The black halo that is seen aroundthe brighter center in (b) is due to perspective effects of the SEM.Inspection with an optical microscope reveals no polymide lip betweenthe top layer of Au and the RDM. The average diameter of the small RDMsis 14±0.28 μm (n=3 cavities). The average diameter of the large RDMs is55.2 μm±0.0 (n=3 cavities). The average diameter of the large RDMs is55.2 μm±0.0 (n+3cavities).

[0055] The depth of the cavities was not measured directly. The smalldiameter of the cavity prevented the use of atomic force microscopy or aprofilometer. To obtain an approximate measure of the depth, thethickness of the polymide layer was measured after patterning using aprofilometer.

[0056] The thickness of the polymide was consistently 4 μm.

[0057] Faradaic Response

[0058] Cyclic voltammetry was used to characterize the electrochemicalresponse of the RDMs. Ru (NH₃)₆ ³⁺ was chosen as a probe because of itswell-established electromechanical properties. A 10 μtm PDM was used forcomparison. One advantage of using disk microelectrodes is that truesteady state currents can be attained. Steady state current is a resultof constant flux to the electrode surface. For static systems it isobtained when the mass transport is dominated by radial diffusion. In asteady state CV, at very slow scan rates where the diffusion layer islarge relative to the size of the electrode, the current of the reversescan should retrace that of the forward scan in a sigmoidal shape. Inthis case, pseudo steady state occurs when the time scale is shortenough and the diffusion layer is thin enough so that transport involvesboth linear and radial diffusion. Cyclic voltammograms in this scan rateare expected to be sigmoidal, with a separation between the forward andreverse currents.

[0059] Graph 1 shows CV responses from the 10 μm PDM (a) and the 14 μm(b) and 55 μm (c) RDMs. All three were obtained in 5.0 mM Ru(NH₃)₆ ³⁺and 0.5 M KNO₃ at 0.1 Vs⁻³. At this scan rate, none of themicroelectrodes exhibit true steady-state behavior. The 10 μm PDM is theclosest, while the 55 μm RDM is the furthest from this behavior. Thedeviation occurs for two reasons. As electrode size increases, thecontribution of linear diffusion to the total flux for a given timescale increases. This is the case in comparing CV responses from the 14and 55 μm RDMs. Secondly, the walls of the cavity prevent radialdiffusion from occurring as long as the diffusion layer is within thecavity. This is demonstrated by comparing CV responses of the 10 μm PDMand the 14 μm RDM.

[0060] The microelectrodes were further investigated to understand theeffects of the cavity. The thickness of the diffusion layer is inverselyproportional to the square of the scan rate and can be approximated byEquation 1,

x=(2Dt)^(½)  (1)

[0061] where x is the thickness of the diffusion layer, D is thediffusion coefficient for Ru(NH₃)₆ ³⁺ (7.8×10⁻⁶ cm²s⁻¹), and t is thetime spent on the reducing side of E⁰, divided by the scan rate. Thescan rate at which the diffusion layer thickness is equal to the depthof the cavity (4 μm) is 58.5 Vs⁻¹.

[0062] Three current regions are defined. At slow scan rates, thecurrent should be independent of scan rate (i.e. steady state). Theequation used to calculate this current is shown below.

i _(ss)=(4πnFC*Dr ²)/(4L+πr)  (2)

[0063] where n is the moles of electrons per mole of analyte involved inthe reaction, F is the Faraday constant (98485 coul*mol electrons⁻¹), C*is the concentration of Ru(NH₃)₆ ³⁺, L is the depth of the cavity, and ris the radius of the disk. A decrease in current relative to planar diskmicroelectrodes (PDMs) of the same radius is expected. For PDMs (whereL=0), the steady state current is.

i ^(ss)=4nFC*Dr  (3)

[0064] At faster scan rates, there will be a transition region whereneither steady state nor linear diffusion models completely apply. Atfast scan rates relative to the depth of the cavity and area of theelectrode, the current should follow the model for linear diffusion andbe proportional to the square root of scan rate, v^(½).

i _(p)=(2.69×10⁵)n ^({fraction (3/2)}) AD ^(½) v ^(½) C*  (4)

[0065] where A is the area of the electrode (πr²).

[0066] A scan rate study was performed to compare our RDMs with thesemodels. The maximum current (i_(max)) was measured from the CVresponses. If no peak is present, then i_(max) is measured to the peak.Charging current was subtracted out. In FIGS. 5 and 6, i_(max) for themicroelectrodes is compared to i_(ss) and i_(p) from Equations 2 and 4as a function of v^(½). The top half (a) of each figure shows scan ratesfrom 0.01 Vs⁻¹ to 10 Vs⁻¹.

[0067] For the 55 μm RDM (L=4 μm), steady state current persists to ascan rate of 0.1 Vs⁻¹. The steady state current for the 55 μm RDM is53.50±0.48 nA. The steady state current predicted by Equation 2 by 34.9nA. The steady state current predicted by Equation 3 is 41.4 nA. Thesteady state current predicted by Equation 3 is 41.4 nA. From thiscomparison, the 55 μm RDMs follow theory for RDMs better than theory forRDMs. The lower calculated current may be due to an inaccurate areadetermination. At faster scan rates, the current increases with scanrate in a fashion like that predicted for Equation 4. At 204 Vs⁻¹, wherethe diffusion layer is thin and the electrodes should follow theory forlinear diffusion (Eq. 5), the maximum current is 784.6±41.2 nA. Thecurrent predicted by Eq. 5 is 1274 nA. This deviation between measuredand predicted current is discussed below.

[0068] For the 14 μm RDM, steady state current persists up until 1 Vs.⁻¹is reached. The maximum current measured at the 14 μm RDMs (5.39±0.96nA) fits the recessed disk model of Eq. 3 (6.10 nA) better than the PDMmodel of Eq. 4 (10.5 nA). For the 14 μm RDM, the curves for i_(p) andi_(ss) vs. ν^(½)cross at 1 Vs⁻¹. Above this scan rate, the currentincreases with increasing scan rate. At 204 Vs⁻¹, the current measuredat the 14 μm RDMs is 35.37±9.51 nA. The current predicted by Eq. 5 is82.58 nA. Again, there is no apparent transition region between steadystate and linear models for this size of RDM.

[0069] At fast scan rates, the current at both the 55 μm and 14 μm RDMsshould be predicted by Equation 4. However, the magnitude for both RDMsis significantly lower. This is due to uncompensated resistance. Theuncompensated resistance, R_(u), was measured with the BAS. Theresistance was found to be 96.6 kΩ for the 55 μm RDM and 209 kΩ for the14 μm RDM. This is higher than that at the 10 μm paM (49 kΩ). Thiseffect has been observed previously for RDMs 15 and for recessedmicroe!ectrode ensembles, however the resistance was not reported to beas high (15-30 kΩ).

[0070] The effect of uncompensated resistance on peak current wasmodeled by computer simulation. The peak current at two scan rates, 50Vs⁻¹ and 204 Vs⁻¹, was measured from simulations at both 0 Ω and either96.6 or 209 kΩ, depending on which was appropriate. A simple ratio wasobtained by dividing the current influenced by uncompensated resistance,i_(R), by the ideal current. A similar ratio was obtained from theexperimental data by dividing the experimentally measured current by thecurrent calculated from Eq. 4 for a given scan rate. For the 55 μm RDM,the experimental ratio at 204 Vs⁻¹ is 0.616. At 50 Vs⁻¹, the ratio is0.784. The values from simulations are 0.726 and 0.817 respectively. Forthe 14 μm RDM, the experimental ratio at 204 Vs⁻¹ is 0.428 and at 50Vs⁻¹ is 0.511. The values from simulations are 0.931 and 0.981,respectively. The deviation of experimental current from planar theoryat fast scan rates can be explained by uncompensated resistance at the55 μm RDMs. The 14 μm RDM cannot be explained completely byuncompensated resistance value given above.

[0071] Charging Current

[0072] Capacitance studies were used to evaluate the quality of theconstruction of the microelectrodes. The charging current was measuredfrom CV in 0.5 M KNO₃ electrolyte. The following equation was used tocalculate the capacitive density.

C=i _(c) /νA  (5)

[0073] where i_(c) is the charging current. Graph 4 shows a log-log plotof the capacitance as a function of scan rate for an Au macroelectrode,14 μm RDM, and 55 μm RDM. Representation of capacitance data in thisform has been used previously to determine the quality of fabrication.Usually, the capacitance is considered to be independent of scan rate.However, even at the macroelectrode this is not the case. The slope forthe RDMs is similar to that of the macroelectrode, indicating that theseal between the insulator and electrode is good, and no cracking hasoccurred. The capacitance values of the 14 and 55 μm RDMs are within afactor of 10 of each other and of the macroelectrode. The smalldifferences may be caused by inaccurate determination of electrode area.

[0074] Convection Studies

[0075] A simple set of experiments were conducted to demonstrate theutility of these microfabricated RDMs in convective systems.Chronoamperometry (CA) was carried out in a 5 mM Ru(NH₃)₆ ³⁺ and 0.5 MKNO₃ solution. Both sizes of recessed disks were compared to a 10 μmPDM.

[0076] Graph 5 is an overlay of the 14 μm RDM and the 10 μm PDM testedin both static and solutions stirred at different rates, and isrepresentative of all repetitions. In the static solution, Graph 5a, thecurrent for the PDM is 7.30 nA, while the current for the RDM is 7.04nA. The steady state current measured in the static solution can be usedto determine the area of each electrode. Using Eq. 3, the effectivediameter of the PDM is 9.70 μm. Using Eq. 2 for the RDM, the effectivediameter is 15.5 μm.

[0077] Graph 5b shows the response in a solution that was stirred at 70rpm. The current measured with the PDM increased to 8.80±0.68 nA and theRDM decreased to 6.65±0.631 nA. Graph 5c shows the response for asolution stirred at 150 RPM. The signal increases to 13.27±0.365 nA forthe 10 μm PDM and the RDM increased to 7.184±0.227 nA. The signal forthe 14 μm.

[0078] The performance of the RDM as an electrochemical detector is bestevaluated by determining the signal-to-noise ratio (SNR). The SNR wascalculated by dividing the average steady state current by the standarddeviation of the steady state current during the reduction step. For the10 μm PDM, the SNR at 70 rpm is 37.6±10.4 and at 150 rpm is 9.89±1.41.The SNR for the 14 μm RDM at 70 rpm is 116±24.7 and at 150 rpm is46.0±12.9. This improvement in SNR is significant even though the depthof the electrode is only 4 μm and the diffusion layer extends wellbeyond the opening.

[0079] A similar set of experiments was carried out for the 55 μm RDM.Unlike the 14 μm RDM, the noise increased with stir rate just as if itwere a PDM. This result is not surprising, although the depth of thecavity is the same. Because of the electrode's larger area, the centeris less protected from convection than the 14 μm RDM. Others havereported the use of RDMs in convective solutions and noise free CA withcavities of approximately 90 μm for RDMs of 25 μm diameter. There is acorrelation between noise and cavity depth with noise disappearingaround a cavity depth of 50 μm for microelectrodes arrays withindividual electrode diameters of 7 μm. The depth-to-diameter value ofboth of these systems is larger (3.6 and 7.1 respectively) than that ofthe microfabricated RDMs (0.29) generated in this work. Despite thisdifference, relatively noise free CA were obtained, however, there isstill noise present that would be further eliminated with a deepercavity.

EXAMPLE 2

[0080] All chemicals were reagent grade and used as received. Aqueoussolutions were prepared with high purity deionized water (Milli-Q). Agold coin (Credit Suisse, 99.99%) and chromium plated tungsten rod (R.O. Mathis) served as sources for thermal evaporation. Silicon wafers(5″, (100)) were donated by the High Density Electronics Center at theUniversity of Arkansas. Potassium nitrate, sulfuric acid, and 30%hydrogen peroxide were obtained from Fisher Scientific. Hexaamineruthenium(III) chloride was purchased from Aldrich Chemical Co. Positivephotoresist (AZ4330RS) and photoresist developer (AZ400K) were obtainedfrom Hoechst-Celanese. Photodefineable polyimide (Pyralin PI-2721) waspurchased from DuPont.

[0081] Array Fabrication

[0082] The fabrication of microcavity electrode arrays was accomplishedthrough the use of photolithographic techniques developed for integratedcircuit technology. A simplified version of this fabrication has beenreported previously. The cavity reported here consists of 5 primarylayers of material. A top down and side view schematic of the CMS areshown in FIG. 4. Layers 1, 3, and 5 are gold, with a Cr adhesion layer,while layers 2 and 4 are polyimide. Layers 1 and 3 serve as themicrodisk and nanoband electrodes. Layer 5 helps maintain the definitionof the cavity and prevent tapering during the etching steps. The arrayswere generated by depositing and patterning each layer of conductor andinsulator. This generated a set of contact lines separated by a sheet ofinsulator for each electrode. The last step in the fabrication was tocreate the cavities and expose the microelectrodes using dry etchingprocedures. Details of fabrication for each layer are listed below.

[0083] Layer 1. Both sides of a single crystal silicon wafer were coatedwith 3 μm of SiO₂ at 250° C. by plasma enhanced chemical vapordeposition (PECVD, Plasmatherm, System VII). Alternatively, the SiO₂could be grown on the wafer by thermal oxidation at 650° C. for 8 hours.This served as an initial passivation layer between the electrodes andsemi-conductive silicon wafer. Layer 1 was patterned using a lift-offprocedure as reported previously, leaving the appropriate pattern asshown in FIG. 4.

[0084] Layers 2 and 4. Wafers were spin-coated with photo-sensitivepolyimide (4 μm). The polyimide film was exposed to 350 nm UV light for12 s through a Karl Suss MA-150 mask aligner to cross-link the polymerleaving a continuous, defect free-insulator film. The polyimide wascured at 150° C. for 30 min, followed by 250° C. for 30 min. The waferwas allowed to cool to room temperature before the fabricationcontinued.

[0085] Layer 3. 15 Å Cr and 500 Å Au were deposited by thermalevaporation. The Au thickness of this layer 132 determines the width (w)of the tubular nanoband electrode. The wafer was spin-coated with 4 μmof photoresist. The photoresist was patterned by exposure through asecond Cr mask. The Au and Cr were etched simultaneously in 50% aquaregia (3 HCl:1 HNO₃:4 H₂O). The absence of the ultra-thin Cr layer wasverified through resistance measurements with a multimeter. Theremaining photoresist was stripped with acetone after the Cr/Au layerhad been etched. After rinsing, the wafer was dried for 30 min at 125°C. prior to coating with polyimide.

[0086] Layer 5. A top layer 5 of Au is essential to producing cavitieswith well-defined, vertical walls. Thermal evaporation was used todeposit 25 Å Cr and 1500 Å Au. Photoresist was deposited and patternedaccording to the procedure for layer 3 using a third Cr mask. The Au andCr were etched with aqua regia as described above. The remainingphotoresist was removed with acetone and the wafer rinsed thoroughlywith deionized water.

[0087] Cavity Formation. Cavities were created using standard dryetching procedures. The wafer was spin-coated with photoresist (6 μm).The photoresist was patterned by exposure to UV light through a fourthCr mask. Layer 5 was etched with RF Ar⁺ sputtering for 5 min and layer 3for 2 min using a 500 V DC potential with constant pressure (30 mT) andflow (50 sccm) of Ar. The polyimide was etched using reactive ionetching (RIE) with a mixture of O₂ (36 sccm) and SF₆ (4 sccm) at 300 mTand 300 W RF power for 13 min.

[0088] Microscopy

[0089] Physical characterization of the microcavities was performed witha combination of light and electron microscopy. A light microscope(Nikon Optiphot) equipped with a calibrated eye piece was used tomeasure the diameter of the cavities. Scanning electron microscopy (SEM,Hitachi S-2300) was used to obtain high resolution images of thecavities and the electrodes inside the cavities. Samples were groundedthrough conductive carbon tabs.

[0090] Electrochemical Characterization

[0091] Electrochemical measurements were made using a BAS-100Bpotentiostat, equipped with a PA-1 pre-amplifier (BioAnalyticalSystems). The system was controlled through a PC with BAS-100W software.A closed three electrode cell with Pt flag auxiliary and Ag/AgCl (sat'dKCl) reference electrode was used for cyclic voltammetry (CV). The 5.0mM Ru(NH₃)₆Cl₃ and 0.5 M KNO₃ solutions were prepared immediately beforeuse. The Ru(NH₃)₆Cl₃/KNO₃ solutions were purged with Ar for 20 min tominimize interference from oxygen reduction during electrochemicalmeasurements. Capacitance was calculated by measuring charging currentfrom cyclic voltammograms collected in 0.5 M KNO₃. The potential wascycled in a region (+400 mV to +100 mV vs Ag/AgCl (sat'd KCl)) where O₂would not interfere with the measurement.

[0092] Scanning Electron Microscopy.

[0093] The initial characterization of the cavity microelectrode systemwas performed with scanning electron microscopy (SEM). A top down viewof a cavity is shown in FIG. 5. The diameter of the cavity is 53 μm. SEMshows a cavity opening that is uniform and smooth. Only the protectiveAu of layer 5 and the disk microelectrode (layer 1) can be seen. A“halo” can be seen in layer 5. This is caused by partial exposure oflayer 5 during the second RF sputtering step resulting in partialremoval (the topmost 100's of Å) of Au where the photoresist has thinnedaround the rim of the cavity.

[0094] Faradaic Response

[0095] Cyclic voltammetry was used to evaluate the electrochemicalresponse of the recessed microdisk (RMD) and tubular nanoband electrode.Graph 6 shows an overlay of cyclic voltammograms (CVs) collected at thetubular nanoband electrode and RDM inside of a single cavity. Bandelectrodes of this size should maintain pseudo-steady state behavior,while the RMDs should be peak-shaped.

[0096] The theory for both microelectrode geometries has been developed.The most commonly reported of the two geometries is the recessed disk,with models for linear and radial diffusion developed. Models describingthe current at nanoband electrodes has also been discussed. Below, wecompare the faradaic response at the electrodes that we have constructedto the current expected based upon the models.

[0097] Recessed Microdisk Electrode. We have discussed theelectrochemical response associated with 4 μm deep RMDs previously. Inthe work presented here, the RMDs are 8 μm deep (instead of 4 μm). Graph7 shows a scan rate study from 0.01-327 Vs⁻¹ for RDMs in a solution of5.0 mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃. Graph 8 is an expanded view of theregion from 0.01-10 Vs⁻¹. At slow scan rates, the current follows radialdiffusion theory. The experimental current (measured at 0.1 Vs⁻¹) is36.65±5.10 nA is greater than the theoretical steady state current(29.32 nA) calculated for recessed disk microelectrodes. The RMDsmaintain steady state behavior to 0.25 Vs⁻¹. At fast scan rates (>0.25Vs⁻¹), the current increases, but does not follow predictions based uponlinear diffusion models. For example, at 204 Vs⁻¹ the i_(max) is566.9±133.7 nA, which is substantially less than the theoreticallycalculated current, 1168 nA. This large deviation is due touncompensated resistance in the system as reported previously.

[0098] Tubular Nanoband Electrodes

[0099] Nanoband electrodes have been studied and an equation (Equation3) derived to predict current as a function of time forchronoamperometry.

i=4nFC*Dl/(ln(64Dt)/w ²)  (1)

[0100] where l is the length of the electrode, w is the width, F isFaraday's constant, D is the diffusion coefficient (7.8×10⁻⁶ cm²s⁻¹), C*is the concentration, and t is the time length of the experiment. Thisequation is based upon hemi-cylindrical diffusion. The length, l, isequal to the circumference (2πr) of the cavity. Unlike the steady stateequation for disk microelectrodes, this equation predicts that thecurrent will be dependent upon scan rate. Secondly, this system willnever generate true steady state current because radial diffusion canonly exist in two dimensions. While this equation was derived for planarband microelectrodes, it can be applied it to tubular bandmicroelectrodes with little deviation from this theory.

[0101] A scan rate study was completed to compare the response of thenanoband electrodes to the theory predicted in Equation 1, and to lineardiffusion models. Graph 9 shows this comparison with 9a showing scanrates from 0.01 Vs⁻¹ to 327 Vs⁻¹, while 13b shows the region from0.01-10 Vs⁻¹. First note the variation of theoretical current with scanrate as predicted. Also, the hemispherical diffusion current is largerthan linear current throughout the scan rate window used in theseexperiments. The measured current, i_(max), follows Equation 1 at slowscan rates (Graph 9b). As the scan rate increases, i_(max) exceeds thepredicted current. This has been reported previously for nano-bandelectrodes and may be the result of a small exposed edge on theelectrode. It is possible that the RIE step used to etch the polyimidewould leave such an edge. This edge would have a restricted diffusionlayer and would not add to the current significantly at slow scan rates.At fast scan rates, the diffusion layer becomes thin enough that currentfrom the exposed lip will add significantly to the current.

[0102] Fabrication Quality

[0103] The quality of the fabrication was evaluated from capacitancevalues. Capacitance was calculated from the charging current obtainedfrom CV experiments in pure electrolyte (0.5 M KNO₃). The capacitancewill have a large dependence upon scan rate if there is poor adhesionbetween the insulator and electrode or if there is cracking in theinsulator around the electrode. The capacitive density was calculatedand compared to a Au macroelectrode. Graph 10 shows a log-log plot ofthree types of electrodes, a macro Au electrode on SiO₂, the RDM, andtubular nanoband electrode. Ideally, the capacitance should beindependent of scan rate, however, the macroelectrode, which has noinsulator, has a dependence. The scan rate dependence of bothmicroelectrodes is close to that for the macroel ectrode and is similarto that seen previously for the RDMs. This indicates that there is agood seal between the insulator and electrode and there is little or nocracking. The magnitude of the capacitance is greater for both of themicroelectrodes This supports the suggestion above that more electrodearea is exposed than the 500 Å wide band. If the area of the tubularnanoband was underestimated, it would make the reported capacitivedensity higher appear higher. This is also true of the disk electrode,however, small variations will not affect the capacitive density to thesame extent.

EXAMPLE 3

[0104] All chemicals were reagent grade and used as received. Aqueoussolutions were prepared with high purity deionized water (Milli-Q, modelRG). A gold coin (Credit Suisse, 99.99%) and a chromium plated tungstenrod (R. D. Mathis) served as sources for thermal evaporation. Siliconwafers (5″, (100)) were donated by the High Density ElectronicsPackaging Facility, University of Arkansas. Potassium nitrate, sulfuricacid, hydrochloric acid, silver nitrate, potassium iodide, sodiumthiosulfate, nitric acid and 30% hydrogen peroxide were purchased fromFisher Scientific. Hexaamine ruthenium(III) chloride and hydroquinonewere obtained from Aldrich Chemical Co. Positive photoresist (AZ4330RS)and photoresist developer (AZ400K) were purchased from Hoechst-Celanese.Polyimide (Pyralin PI-2721, DuPont) was purchased from DuPont.

[0105] Cavity Microelectrode Construction.

[0106] The fabrication of the cavity electrode system (CES) has beendescribed previously. In brief, the CES is made by depositing andpatterning alternating layers of Au and polyimide on an oxidized Siwafer, with a total of five layers. A schematic of the CES is shown inFIG. 6. Layers 1, 3, and 5 are Au, with a Cr adhesion layer. Layers 2and 4 are a polymeric insulator, polyimide. After these layers have beendeposited and patterned, a cavity is etched through the top 4 layers,exposing a 500 Å wide tubular nanoband electrode (TNE) and a recessedmicrodisk electrode (RMD). Two diameters of cavity are reported here, 13μm and 53 μm. Both cavities are 8 μm deep.

[0107] Electrochemical Measurements

[0108] A BAS-100B potentiostat and PA-1 preamplifier controlled withBAS-1 a 100W electrochemical software were used to perform cyclicvoltammetry (CV) and chronoamperometry (CA). For characterizationexperiments, a Pt flag auxiliary and macro Ag/AgCl (sat'd KCl) referenceelectrode were used to complete the three electrode system. Stirringstudies involved CA and were performed on a Coming PC-320 stir platewith a ½″ magnetic stir bar (Fisher Scientific) The cell volume was 40mL and was not purged prior to CA. The rotation rate was determined bycounting the rotations of the stir bar over a given time period.

[0109] Microreference Formation

[0110] Formation of a Ag/Agi pseudoreference microelectrode wasaccomplished following a procedure developed by Bratten et al and wellknown in the art. Ag was deposited for 1 s on the TNE at −0.5 V versus aPt flag from a solution containing the complex ion [AgI₂]⁻K⁺. Thecomplex ion was obtained in a solution of 0.1 M AgNO₃, 1 M KI and 0.25mM Na₂S₂O₃. The Ag was oxidized in saturated KI for 0.5 s at +0.5 Vversus a Pt flag. After formation of the reference electrode, theelectrodes were rinsed thoroughly with deionized water, dried, andstored in a covered vial. Stability of the Ag/AgI pseudoreference wasdetermined using cyclic voltammetry in 5.0 mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃solution. The E^(o) potential was compared to the potential determinedusing a macro Ag/AgCl (sat'd KCl) reference electrode.

[0111] Small Volume Analysis

[0112] The application of the CES to measurement of electroactivespecies in small volumes was accomplished using the Ag/AgIpsuedoreference and layer 5 as the auxiliary electrode. A small volumeof solution was placed on the cavity using an automatic pipette. Twosolutions were tested using the CES. A 5.0 mM Ru(NH₃)₆ ³⁺/0.5 M KNO₃solution was used as the model system because of its well understoodproperties. The second analyte tested was hydroquinone. A 4.0 mMsolution in 0.5 M KNO₃ solution buffered to pH 6.60 with 0.05 Mphosphate buffer was analyzed.

[0113] Convection Studies

[0114] Convection studies were carried out using both diameters of CES.The internal reference and auxiliary electrodes were used. The protocolused for testing electrodes in convective systems has been reportedpreviously. In brief, a solution of either 5.7 mM Ru(NH₃)₆ ³⁺ and 0.5 MKNO₃ or 1.0 mM hydroquinone in pH 6.60 phosphate (0.05 M) buffer wasplaced in a cell containing a ½″ magnetic stir bar. The electrodes weretested in both static solution and solution stirred at either 70 or 150rotations per minute (rpm). Chronoamperometry (CA) was used to determinethe effect of convection on faradaic current. For the Ru(NH₃)₆ ³⁺, thepotential was stepped from +0.2 V to −0.4 V vs Ag/AgI for 5 s. For thehydroquinone, the potential was stepped from +0.2 V to +0.75 V vs Ag/AgIfor 5 s.

[0115] Results and Discussion

[0116] The cavity electrode system (CES) was evaluated using cyclicvoltammetry (CV) in Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃ solution. Cavities of twodifferent diameters. 53 and 13 μm, were compared with models presentedfor each electrode geometry. The CES contains two electrodes, a tubularnanoband electrode (TNE) and a recessed disk microelectrode (RMD) in theconfiguration shown in FIG. 6. An exhaustive comparison to models forboth electrodes in a 53 μm diameter cavities has already been reported.Graph 11 compares CVs from an RMD and TNE in both 53 (a, b) and 13 μm(c, d) cavities.

[0117] At slow scan rates, the RMD in a 53 μm cavity has been shownpreviously to follow the model for radial diffusion to a planarmicrodisk electrode (PMD), while the RMD in a 13 μm cavity follows themodel for radial diffusion to a RMD. The steady state current measuredfor the 53 μm RMD (Graph 11a) is 39.5±2.93 nA which matches closely withthe predicted current (39.9 nA). The steady state current for the 13 μmRMD (Graph 11c) is 2.13±0.46 nA which is less than the predicted current(3.81 nA).

[0118] The electrochemical behavior of the TNE in a 53 μm diametercavity has been described and found to follow models for radialdiffusion to a band electrode. The comparison of a TNE to models forradial diffusion to a band electrode in a 13 μm cavity has not beenreported. At 0.1 Vs⁻¹, the experimental current for the TNE in a 53 μmcavity is 25.8±4.2 nA, which matches with the predicted current (16.2nA). For the TNE in a 13 μm cavity, the experimental current is6.31±0.28 nA, which is greater than the predicted current (3.98 nA).

[0119] Formation and Stability of Ag/AgI Pseudoreference.

[0120] The ability to make accurate potential measurements in smallvolumes of samples requires the presence of a reference electrode.Others have reported the use of a Ag/AgI pseudoreference microelectrodefor small volume measurements. Ag/AgI was deposited on the TNE and CV in5.0 mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃ was used to characterize the system.Layer 5 was used as the auxiliary electrode to complete theelectrochemical system. The standard reduction potential of AgI is−0.1519 V vs the normal hydrogen electrode (NHE) as compared to AgCl at+0.2223 V vs NHE. The experimentally determined reduction potential ofRu(NH₃)₆ ³⁺ vs Ag/AgCl (sat'd KCl) is −0.16±0.002 V, but −0.053±0.016 Vvs the micro-Ag/AgI pseudoreference. The redox potential of Ru(NH₃)₆ ³⁺vs Ag/AgI, predicted by standard reduction potentials, should be +0.222V. The 0.275 V shift in reference potential is probably due to the lackof I⁻ in the supporting electrolyte solution.

[0121] After determining that the reference potential of the Ag/AgIpseudoreference electrode was more positive than predicted, a study wasdone to determine if the electrode was stable over time with multipleexperiments. A simple study was done to determine the reliability of theAg/AgI electrodes from run to run. A 53 μm cavity was placed in 5.0 mMRu(NH₃)₆ ³⁺ and 0.5 M KNO₃ solution and 10 CVs collected at 0.5 Vs⁻¹,allowing the system to go to open circuit between each cycle. The 53 μmcavity was chosen because the RMD draws more current than the TNE andtherefore, the microreference in the 53 μm cavity should be less stablethan the 13 μm CES. Graph 12 shows an overlay of the first, fifth, andtenth cycles. The E^(o) potential does not change between runsindicating that the pseudoreference electrode is stable in the absenceof supporting I⁻ ions.

[0122] Small Volume Analysis

[0123] The application of the CES to small volume analysis was firstdemonstrated with Ru(NH₃)₆ ³⁺ as the analyte. Graph 13 shows an overlayof CVs collected with an RMD in a 13 μm CES, in two different volumes,10 and 1 μL. The response of the working electrode in these two volumesis essentially identical. The experimental current is 2.89±0.53 nA forthe 10 μL sample and 2.67±0.46 nA for the 1 μL sample. E^(o) for the 10μL sample is −0.086±0.034 V and for the 1 μL sample is −0.123±0.063 V.

[0124] The second small volume system tested was hydroquinone.Hyrdoquinone is a common moiety in pharmaceutical and biologicalcompounds. The analysis of the 10 and 1 μL samples of hydroquinone isdemonstrated in Graph 14. The current for the two samples is withinerror, 2.91±0.84 nA for the 10 μL and 2.58±0.72 nA for the 1 μL.

[0125] Convection Studies

[0126] Both diameters of CES were tested in stirred solutions todemonstrate the advantageous nature of RMDs in convective systems. RMDsof similar diameter with a 4 μm depth have been tested previously. RMDswith a 14 μm diameter were found to provide a 4 fold improvement insignal-to-noise ratio (SNR) over planar microdisk electrodes (PMOs).

[0127] To obtain an accurate measure of the effects of convection on thefaradaic current, chronoamperometry (CA) was used. The potential wasstepped from a region of non-faradaic current transfer to a region ofeither oxidation (hydroquinone) or reduction (Ru(NH₃)₆ ³⁺). No datasmoothing was employed and a lowpass filter with cutoff frequency of2500 Hz was used to minimize electrical noise. Graph 15 shows theoxidation step for hydroquinone in pH 7.0 phosphate buffer with a 53 μmCES in either static solution or solution stirred at 70 or 150rotations-per-minute (rpm). The potential was stepped from 0.0 V to +0.6V vs Ag/AgI for 5 s. The signal-to-noise ratio (SNR) was calculated byfirst determining the steady state current between the 1st and 5th s ofthe potential step. The current was then divided by the standarddeviation of the data during the same time duration. The noise presentin the static solution is due to electrical noise. The SNR in staticsolution is 9.85±1.8. The SNR in a solution stirred at 70 rpm is8.37±0.86 and at 150 rpm is 8.99±2.20. All three of these fall withinerror of each other. Graph 16 shows a reduction step for Ru(NH₃)₆ ³⁺ ata 13 μm CES for a static solution and solutions stirred at 70 and 150rpm. The SNR was calculated following the procedure discussed above. Forthe static solution, the SNR is 5.30±2.7. For the solution stirred at 70rpm, 5.60±2.44 and at 150 rpm, 5.93±2.39. Again, the SNRs are withinerror of each other independent of stirring.

EXAMPLE 4

[0128] Materials. Hexadecanethiol (C₁₆SH, Aldrich) solutions wereprepared as described previously except with rigorous exclusion of airsThe C₁₆SH was filtered through alumina (Brockman, neutral, activity I)prior to the preparation of fresh derivatization solutions. Absoluteethanol (100%, Millennium Petrochemical), magnesium nitrate hexahydrate(99.995%, Aldrich), potassium nitrate (99.999%, Acros, N.J.), sulfuricacid (Fisher), methanol (HPLC grade, Fisher); CHCl₃ (HPLC grade,Fisher), and hydrogen peroxide (30%, Fisher) were used as received.Zero-grade argon and nitrogen (Air Products) were used to purge thesolutions and maintain inert atmosphere in a glove bag (Instruments forResearch and Industry, Cheltenham, Pa.) during surface derivatization.Milli-Q RG (Millipore) filtered water was used for all aqueous solutionsand rinsing. Dimyristoyl phosphatidylcholine (DMPC), obtained fromAvanti Polar Lipids, and gramicidin D (gD), obtained from Sigma ChemicalCo., were used without further purification. Gramicidin D is a mixtureof gramicidin A, B, and C, of which g.A is the major component.

[0129] Substrate Preparation. Gold substrates were prepared in anEdwards E306A thermal evaporator. Approximately 50 Å of chromium fromchromium-plated tungsten rods (R. D. Mathis) was deposited as anadhesion layer, followed by 2000-2500 Å of gold (Canadian Maple Leaf,99.9% or Credit Suisse, 99.99%) onto clean silicon wafers (Silicon QuestInternational). Immediately prior to modification by SAMs, goldsubstrates were cleaned in piranha solution, which is a 3:7 solution of30% H₂O₂ and conc. H₂SO₄ Caution: This solution is very corrosive andmust be handled with extreme care. Substrates were then rinsedthoroughly in DI water (Milli-Q RG).

[0130] Monolayer and Bilayer Preparations.

[0131] Self-assembled monolayers were formed by soaking Au substrates inderivatizing solutions of 1 mM C₁₆SH or 0.1 mM gD in 1 mM C₁₆SH (inethanol) for 12 h. Electrodes were removed from solution and rinsed withAl-purged ethanol prior to performing experiments. Solution preparationand monolayer formation were performed in an Ar-purged glove-bag tominimize air-oxidation of thiolates. Samples were only exposed to airwhen placed in the dry, CO₂-free air-purged (Balston) PM-FTIRRAS chamberor when transferring to the XPS sample chamber.

[0132] Vesicle suspensions of DMPC and DMPC+gD (mole ratio: 28 DMPC to 1gD) used for bilayer formations were prepared according to publishedmethods. To 100 μg of dried gD (from 50 μM gD/CH₃OH stock solution) 1.5μmol DMPC (from 1 mg/10 μL DMPC/CH₃OH stock), was cosolubilized alongwith 90 μL CH₃OH and 100 μL CHCl₃. The solution was mixed and theresulting suspension was dried under vacuum overnight to remove theorganic solvent. The dried gD/lipid mixtures were resuspended in 500 μLof Ar-purged DI water and sonicated for 2 h at 55° C. The gramicidinconcentrations in vesicle solutions were determined by measuring theabsorbance at 280 nm (ε=20840 M⁻¹ cm⁻¹) using an HP 8452A Diode Arrayspectrophotometer. Typical gD concentrations are from 0.9-1.4 mM. Theconformation of gD in vesicles was determined by circular dichroism (CD)measurements that were obtained at room temperature using a JASCO 710Aspectrometer. The characteristic positive peaks at 218-220 nm and235-236 nm, a positive minimum at 229-230 nm and a negative ellipticitybelow 208 nm, indicate that gA exists in a β channel conformation in theDMPC vesicles.

[0133] Electrochemical Measurements.

[0134] The electrochemical cell consisted of a standard three-electrodesystem. The reference was a Ag/AgCl (saturated KCl) electrode. Aplatinum flag electrode served as the auxiliary electrode. Immediatelyprior to electrochemical experiments, solutions were purged thoroughlyin a closed cell to minimize the presence of oxygen. Capacitance, C, ofalkanethiol monolayers and hybrid blamers were determined by twodifferent methods, cyclic voltammetry and AC impedance. Cyclicvoltammetry was performed using a computer-interfaced potentiostat(BioAnalytical Systems, 100B with 100W software), and was previouslydescribed. Cyclic voltammetry was performed over two different potentialranges: between 450 mV and 300 mV, and between 100 mV and −100 mV, in0.1 M KNO₃ (reagent grade and 99.999%) and in 0.1 M Mg(NO₃)₂ (reagentgrade and 99.995%). The equation C=i_(c)/νA was used to calculate C. Thei_(c) is the cathodic or anodic charging current at a given potential(400 mV or 0.0 mV), ν is the scan rate, and A is the electrode area.

[0135] AC impedance measurements were made using a EG&G PAR M273Apotentiostat, M388 Electrochemical Impedance Systems software, and EG&GPAR M5210 lock-in amplifier. A sinusoidal ac signal was applied atfrequencies between 10 and 64,000 Hz. Measurements were made in 0.1 MKNO₃ (99.999%) and 0.1 M Mg(NO₃)₂ (99.995%) with a 10 mV amplitude at0.0 V vs Ag/AgCl (sat'd KCl) reference. Errors that are reported intables or as bars in the plots represent one standard deviation.

[0136] Ellipsometry. The procedure for measuring film thickness wasdescribed previously. A Rudolph Research Model 43603 ellipsometerequipped with a 5 mW helium-neon laser light source (632.8 nm) and witha 700 angle of incidence was used to measure the monolayer and bilayerthicknesses. Six measurements were obtained at various sites on eachfreshly cleaned Au substrate and subsequently modified. The change inpolarization state and phase change of the electric field associatedwith the light beam were determined. The averages of these measurementswere used to calculate the film thickness on each modified substrate. Arefractive index of 1.45 for hydrocarbon layers on gold was assumed.Errors that are reported in tables represent one standard deviation.

[0137] PM-FTIRRAS Spectroscopy. Infrared spectra of the modifiedelectrodes were obtained with a Mattson Research Series polarizationmodulation Fourier-transform infrared spectrometer (PM-FTIRRAS). TheFTIR beam was focused onto the sample at an incident angle of 77°. Thebeam was polarized and passed through a ZnSe Series II (Hinds)photoelastic modulator (PEM) operating at 37 kHz before reaching thesample. The reflected beam was detected using a liquid nitrogen cooledHgCdTe detector. Spectra were taken with 2 cm⁻¹ resolution, as indicatedin figure captions. PM-FTIRRAS spectra were normalized by fitting thedifferential reflectance spectra to ninth order polynomial backgroundsusing FitIT, curve fitting software (Mattson). After curve fitting,spectra were truncated and converted to absorbance using WmFirst macrowritten in-house under the specifications of Mattson.26.

[0138] X-ray Photoelectron Spectroscopy. The XPS spectra were obtainedusing Kratos Axis HSi spectrometer equipped with a monochromated AlKα.source, 180° hemispherical analyzer and 5-channeltron detectors. Thepressure in the analytical chamber during analysis was about 10⁻⁹ Torr.The sampling area was 0.4 mm×0.7 mm. Gold and modified-gold samples wereattached to the sample holder with grounding screws. The selectedregions of the spectra were normalized against the Au(4f_(7/2)) peakheight. For the gD and DMPC powder samples, a thick coating of finelyground powders of each samples were pressed into a double-sided tape andattached to a sample plate made from a Si wafer. Charge compensationwith an electron flood gun was only used when analyzing the powdersamples. All powder sample spectra were charge-corrected to bring theC(1s) hydrocarbon peak energy to 285.0 eV. The magnitude of the chargecorrection was 1.5

[0139] Characterization with ellipsometry. We have previously usedellipsometry to characterize gold surfaces that have been modified byfusion of phospholipid vesicles of DMPC with self-assembled monolayersof hexadecanethiol. Those results, which are duplicated in Table I,demonstrate that this procedure forms blamers reproducibly. The firstlayer consists of a monolayer of 22.2±1.5 Å of hexadecanethiol (or19.6±2.0 Å if formed in the presence of gD), and the second layer ofphospholipids, which depending on the first layer and whether gA ispresent, increases the thickness by an additional 21 to 25 Å. Here, wereport a more thorough analysis of these layers using ellipsometry,including exploration of the extent of vesicle physisorption andcontribution of gD to total thickness of each layer. These results arealso reported in Table I.

[0140] The results for the film formed on a bare gold sample that hasbeen placed in a suspension of DMPC vesicles is 19.5±11.2 A. The averagethickness of physisorbed DMPC on gold is lower than that of a DMPC layerformed at a C₁₆SH SAM. This could be due to lower coverage and lessorder. In addition, the large standard deviation implies that the DMPCphysisorbed to gold is not uniformly distributed. Thus, it appears thatthe SAM is necessary to initiate reproducible and specific fusion ofDMPC vesicles with the surface.

[0141] Based on surface coverage studies, the presence of gD in thethiol solution during assembly of C₁₆SH onto gold appears to cause adecrease in the total C₁₆SH that attaches to the surface. However, theaverage thickness for a SAM, formed with a gD in solution, is lower, butwithin error at 95% confidence of that of a pure C₁₆SH SAM. To furtherinvestigate the effect of gD on the modified surfaces, we obtainedadditional ellipsometry results for hybrid blamers in which gD waspresent during formation of only one of the two layers, either in thefirst layer, gD+C₁₆SH, or in the second layer, DMPC+gD. The thicknessesof these two types of hybrid blamers are significantly different at the95% confidence level. The presence of gD in the formation of the firstlayer has a significant impact on the structure of the bilayer. This isconsistent with our previous surface coverage results and ellipsometryof the monolayers. However, when gD is present in forming both the firstand second layers (C₁₆SH+gD/DMPC+gD) the total film thickness is thegreatest of all of these combinations. This seems to indicate thatDMPC+gD vesicles can fill in gaps or defects in the underlying layerbetter than DMPC alone. This may be due to the ability of gD to transferfrom the phospholipid layer to the SAM layer, as it does in planarphospholipid blamers.

[0142] Although ellipsometry serves as a sensitive measure of thickness,there remains unanswered questions about the composition and structureof the layers on the surface, especially in the presence of gD. Inaddition, the calculation of thickness from the ellipsometrymeasurements involves the assumption that the refractive index is thesame for C₁₆SH, phospholipids, and gA. Thus, it is essential that othertechniques be used to further elucidate the structure and verify thevalidity of the ellipsometry results.

[0143] Characterization with PM-FI'IRRAS. There has been a substantialnumber of structural analyses on phospholipid films performed in air byvarious infrared techniques. Many of these studies involve attenuatedtotal reflectance (ATR) IR, in which the phospholipid films aredeposited onto a substrate using LB-deposition methods or by castingfilms onto ATR plates, (not via vesicle fusion). One reason that vesiclefusion has not been used in ATR-IR is that the primer layer ofalkanethiols do not covalently attach to the substrate material, whichis usually Ge, ZnSe, or Si. Thin films of metal can be deposited ontothe substrate material. However, the metal film must be partiallytransparent, and thus, may not be representative of the surfacemorphology of bulk metal. films. External reflectance, such asPM-FTIRRAS, eliminates such special requirements for the substrate.Thus, PM-FTIRRAS was used here to evaluate the structure and compositionof the modified surfaces. Several IR bands of gA have differentfrequencies from DMPC and from C₁₆SH, so that we can monitorcompositional variation in the hybrid blamers. Table n summarizes thepeak positions and assignments for PM-FTIRRAS spectra for filmscontaining different combinations of C₁₆SH, gD, and DMPC, and fortransmission FTIR spectra of gD and DMPC in a KBr pellet.

[0144] Graph 17 shows representative IR spectra for C₁₆SH SAMs,C₁₆SH/DMPC, blamers, and DMPC dispersed in KBr. In the spectrum of aC₁₆SH SAM (Graph 17a), the −0.0012 absorbance for the Vu CH₂ band isclose to values reported for a monolayer coverage for C₁₆SH on gold(−0.0014 AU). Other points to note in FIG. 1a are the hydrocarbon peakpositions. The bands at 2964 cm⁻¹ and 2877 cm⁻¹ are assigned to the CH₃asymmetric in-plane CH-stretching modes, respectively.

[0145] Those at 2918 and 2850 cm⁻¹ are assigned to the asymmetric andsymmetric CH₂ modes, respectively. These values are in good agreement,within ±1 cm⁻¹, of values reported in the literature for long-chainmeasurements.

[0146] The spectrum for a C₁₆SH/DMPC hybrid bilayer is shown in Graph17b. The absorbance values of the CH-stretching bands are essentiallydoubled from that of the SAM. We interpret this to mean that a bilayerhas formed. These data are consistent with the ellipsometrymeasurements.

[0147] The DMPC has 12 methylene carbons in the alkyl chains, not 15,and thus we would expect to see an 80% increase in the absorbance of theCH₂ bands, if no significant changes in orientation occur. The actualincrease is 78.7±3.2% AU for the CH₂ ν_(as) band. We would expect adoubling of the CH₃ absorbances. The actual increase is 201.6±33.3% AUfor the CH₃ ν_(as) band. Other evidence for the presence ofphospholipids includes ester carbonyls at 1739 cm⁻¹ and the asymmetricPO₂ ⁻ and symmetric PO₂ ⁻ stretches at 1260 cm⁻¹ and 1100 cm⁻¹,respectively. The relative magnitude of the carbonyl stretch varies fromsample to sample by ±0.0006 AU, which might be an indicator of smallvariations in the orientation of the head group. The only frequencies ofthe CH-stretching modes for the C₁₆SH/DMPC blamers that differ fromthose for the C₁₆SH SAM are the CH₃ asymmetric (2962 cm⁻¹) and the CH₂asymmetric (2917 cm⁻¹) modes. However, these are within the resolutionof our measurements and might not be significant. In addition, if amonolayer consists of alkyl chains in all-trans configuration, theν_(as) CH₂ band will occur at 2918 cm⁻¹, as compared to 2925 cm⁻¹ forfilms with a more disordered, liquid-like structure. Overall, ourresults indicate that the structure of the hydrocarbon chains in theDMPC layer are structurally similar to those in the SAM.

[0148] Graph 17c shows the transmission IR spectra for DMPC in a KBrpellet for comparison. The ester carbonyl peak for DMPC in the KBrpellet (1745 cm⁻¹) is lower by 3 cm⁻¹ than that in the hybrid bilayer.Those bands for the PO₂ ⁻ asymmetric stretching mode at 1252 cm⁻¹, andthe PO₂ ⁻ symmetric stretching mode, observed at 1092 cm⁻¹, are lowerfor DMPC in the KBr pellet than that in the hybrid bilayer by 8 cm⁻¹.The structure of DMPC in the hybrid blamers is clearly different than inthe powder form.

[0149] Graph 18 shows the PM-FTIRRAS spectra for the C₁₆SH+gD monolayer,C₁₆SH+gD/DMPC+gD bilayer, and the transmission spectrum of gD in a KBrpellet. In the spectrum for ethanol-rinsed C₁₆SH+gD (graph 18a), thereare no characteristic IR bands for gA, which are the Amide I (C═Ostretch) the Amide II (coupled CN stretch and NH bending), and Amide A(NH stretch) bands. Thus, it appears that gD is not present insufficient concentrations in the SAMs formed from C₁₆SH+gD to bedetected. In addition, the absorbance for the CH-stretching modes isonly approximately 90% of that for a SAM formed from C₁₆SH alone.Interestingly, the frequencies of the CH-stretching modes are identicalto those for the pure C₁₆SH SAM. This indicates that any changes in themonolayer structure due to assembly in the presence of gD are notdetectable with PM-FTIRRAS. We have included a PM-FTIRRAS spectrum for afilm formed from a C₁₆SH+gD, followed by rinsing with water, instead ofethanol (Graph 19). Our previous work demonstrated that anirreproducible number of multilayers form after a water rinse, becausewater does not solvate gA or C₁₆SH. A comparison of this spectrum andGraph 18a provides two main points. First, it lends credibility to thefact that the IR bands for gA are easily detectable and comparable inabsorbance to those of C₁₆SH for films when in a 1:10 ratio (as in thesolution). Second, it supports our earlier work that had only indirectlysuggested that gD must be removed from the SAM during the ethanol rinse,perhaps leaving behind gaps or defects in that layer. The water rinse,however, causes multilayer formation, evidenced by a 7 times increase inthe absorbance value in the CH-stretching region from that for anethanol-rinsed sample.

[0150] Graph 18b shows that in a bilayer of C₁₆SH+gD/DMPC+gD, theabsorbance bands in the CH-stretching region is approximately two timesthe absorbance of monolayers. Gramicidin is present in the film thistime, as demonstrated by the prominent Amide I peak at 1660 cm⁻¹, AmideII peak at 1546 cm⁻¹ and the weak, broad Amide A band at 3288 cm⁻¹. Alsopresent is the sharp band at 1739 cm⁻¹ for the ester carbonyl of DMPC,as well as the peaks assigned to asymmetric and symmetric PO₂ ⁻stretching modes. The absorbance of ester carbonyl band is higher thanthat for the C₁₆SH/DMPC blamers. This may be an indication that there issome orientational change in the DMPC head group relative to the surfacein the presence of gD. The magnitude of this band does vary, however,from bilayer to bilayer by as much as 15% AU relative standarddeviation.

[0151] Graph 18c shows the transmission IR obtained from gD in a KBrpellet. The characteristic bands are the Amide A at 3278 cm⁻¹, the AmideI at 1637 cm⁻¹, and the Amide II 1536 cm⁻¹. These are lower in frequencyby 10 cm⁻¹, 23 cm⁻¹, and 10 cm⁻¹, respectively than the correspondingbands in the hybrid blamers containing gD. It has been reported that thefrequency shift of the Amide I band suggests a change in environmentsaround the gramicidin molecules and/or a conformational change caused bythe introduction of gramicidin into the lipid layer.

[0152] XPS Analysis. XPS measurements were also used to extractinformation regarding surface composition for mono layers and blamers.This method has a sensitivity of 0.01-0.3% of the elemental compositionof the surface, and thus provides a complementary analysis method to IR,which is about a factor of 10 less sensitive for surface analysis. XPSspectra of O(1s), N(1s), C(1s), and S(2p) regions of C₁₆SH SAMs werecompared with those of SAMs formed from derivatizing solutionscontaining gD. The results of the O(1s), N(1s), C(1s), and S(2p) regionsof each ethanol-rinsed C₁₆SH, ethanol-rinsed C₁₆SH+gD, and water-rinsedC₁₆SH+gD self-assembled films are shown in FIGS. 25a, 25 b, and 25 c,respectively.

[0153] XPS analysis of the C16SH SAM (Graph 20a) shows a C(1s) at 284.9eV for the aliphatic hydrocarbons. This value is comparable to reportedliterature value of 284.7 eV. Also the S(2p) doublet at 161.9 eV and163.0 eV are consistent with reported values for thiolates bound togold.

[0154] SAMs formed in the presence of gD, followed by an ethanol rinse,yield XPS spectra (Graph 20b) with peaks for C(1s) and S(2p) that aresimilar to those for the C₁₆SH SAM alone. There are no new peaks forN(1s), 0(15), or for C(1s) that correspond to gD. Thus, these data areconsistent with the PM-FTIRRAS data that show that the gD is removedfrom the surface during an ethanol rinse.

[0155] An XPS analysis of water-rinsed samples of C₁₆SH+gD is shown inGraph 20c. The strong O(1s) peak at 531.6 eV and the N(1s) peak at 400.5eV indicate the presence of the amide groups in gD. Also present is theshoulder at 286.6 eV representing the C(1s) emissions from the amidecarbonyl groups. These results are consistent with the PM-FTIRRAS dataand previous work.

[0156] Graph 21a shows the O(15), N(1s), and C(1s) XPS spectra of gDpowder. The O(1s) peak for the gD powder appears at 531.4 eV. This valueis in good agreement with values reported for poly(L-amino acids),531.5-532.1 eV. As expected, we observe more than one C(1s) peak. Theamide carbons (C—N and C═O) of the peptide backbone appear as a broadshoulder with peaks at 288.2 eV and 278.0 eV. These values are inagreement with literature values to within 0.4 eV and 0.1 eV for the C—Nand C═O C(1s) regions, respectively. The N(1s) peak is observed for theamide groups in gD at 399.5 eV.

[0157] Graph 21b shows the O(15), N(1s), C(1s), and P(2p) XPS spectrafor DMPC powder. The three chemical states that give C(1s) signals forphospholipids are those assigned to C—C and C—H at binding energy of285.5 eV, C—O at 286.6 eV and O═C—0 at 288.8 eV. The C(1s) region of theDMPC powder appears as a broad peak ranging from 286 eV to 289 eV. Alsopresent is the N(1s) peak at 402.2 eV for the nitrogen of the lipid headgroup as well as the very weak and broad P(2p) peak at 134.2 eV.

[0158] The XPS analysis performed on the hybrid blamers with and withoutgD yield irreproducible results. The presence of N(1s) and P(2p) peakswas sporadic from sample to sample. The PM-FTIRRAS spectra before andafter XPS analysis of the same sample were identical. At this time, itis not clear why the XPS spectra are not as reproducible as the othercharacterization of these samples. Perhaps sample damage in the smallX-ray irradiated area occurs and thus affect the XPS results, but is notsufficient to perturb enough of the sample to affect the IR analysis,which covers an area −110 times larger.

[0159] Capacitance in KNO₃ and Mg(NO₃)₂ Electrolyte. Electrochemicalanalysis provides additional information about the barrier properties ofthe modified surfaces to solution species. The capacitance at anelectrode surface is highly dependent on composition of the electrolyte,solvent, nature of the electrode surface, and applied potential. Thereare numerous reports that draw general conclusions about SAMs andphospholipid layers based on AC impedance and other capacitancemeasurements such as cyclic voltammetry in simple electrolytes. However,many reports do not provide supporting analyses from other techniques.Here, we correlate our spectroscopic data to capacitance determinationsand compare to the conclusions found in the literature. Potassium- andmagnesium-containing nitrate salts were chosen to evaluate the relativepermeation of mono- and dications through the films, respectively.Capacitance values were obtained by CV and ac impedance and are reportedin Tables III and IV.

[0160] Overall, the capacitance data seem to be consistent with thespectroscopic data. Specifically, the decrease in capacitance frommonolayers to blamers is close to 50%, which is expected if the bilayeris double the thickness of the monolayer and if the molecules in the twolayers are identical. There is a more dramatic change in capacitance forthe gD-containing films (46% for KNO₃ and 54% for Mg(NO₃)₂ than forthose without gD (40% for KNO₃ and 48% for Mg(NO₃)₂. This is consistentwith ellipsometry that shows a 128% and 98% increase in thickness forblamers with and without gD, respectively. Likewise, when IR indicateslower coverage of CH-containing molecules, the capacitance in KNO₃increases, such as when comparing SAMs of C₁₆SH to SAMs of C₁₆SH+gD orwhen comparing blamers of C₁₆SH/DMPC to blamers of C₁₆SH+gD/DMPC+gD.

[0161] The type of electrolyte can play a significant role. Ifspectroscopic analysis are unavailable, interpretation of capacitancemay be difficult. However, determination of capacitance in differentelectrolytes can provide valuable insights into film structure andcomposition. For example, the relative capacitance trends in Mg(NO₃)₂,for films with and without gD, are opposite those of KNO₃ Possiblereasons for this are described further below.

[0162] Capacitance values determined from the charging current in CV(100 mV/s), measured at 0.4 V, are shown in Table III for all versionsof the modified electrodes. There are two sets of data. The first set ofvalues was obtained in reagent grade 0.1 M KNO₃ and 0.1 M Mg(NO₃)₂ (fromMg(NO₃)₂*6 H₂O) and for samples which were not protected from air duringtransfers to and from the electrochemical cell. These were reportedpreviously. It has been suggested that SAMs air oxidize upon exposure toair, which may change the capacitance of the layers. Also, theimpurities in the electrolytes used previously, and in particular thoseof the Mg-containing salt, may have led to capacitance values that couldnot be accurately predicted. Thus, a second set of capacitance values(Table III) were obtained in solutions prepared from higher puritysalts, KNO₃ (99.999%) and Mg(NO₃)₂*6 H₂O (99.995%), and for whichsamples had not been exposed to air at any point in the preparation orelectrochemical analysis steps.

[0163] The first and second set of C values obtained in KNO3 are withinerror for a given film composition. In both cases, higher capacitance isobtained in KNO₃ when gD is present in the derivatizing solutions toform the monolayers (34-38%) and blamers (14-0.24%). In Mg(NO₃)₂ thereis a decrease in capacitance from C₁₆ SH SAMs to C₁₆SH+gD SAMs. Thatdecrease is 22% and 20% for the first set and second set, respectively.For blamers, the decrease in capacitance in Mg(NO3)2 when gD is presentis 26% and 17%, respectively. Although the absolute capacitance valuesfor the Mg(NO3h electrolyte in the second set are lower than for thefirst set, the trends observed in the absence and presence of gD aresimilar and reproducible.

[0164] The monolayer-modified samples show a similar trend incapacitance with gD as the blamers. Yet, it is known, based on thespectroscopic evidence described above, that the C16SH+gD monolayer doesnot contain significant amounts of gD. Thus, for that modified surface,one cannot propose that the selective permeation is based on ion channelconformation. Also, such structural and compositional changes maycontribute to the selective-ion effect for blamers with and without gD.Based on these results, it appears that the ion-selectivity of the filmswith gD are governed by film composition, not ion-channel conformation.

[0165] The capacitance values in Table III are higher than thosereported by others for SAM and phospholipid modified electrodes. Thecapacitance for the C₁₆SH/DMPC bilayer in purified 0.1 M KNO₃ is about2.6 times greater than those reported for C₁₈SH/POPC blamers on Au in0.010 M KCl and −1.6 times greater than for C₁₀SH/POPC bilayer in TBS.

[0166] We believe that those differences are due to the manner in whichthe capacitance was determined, not because of large differences insample preparation. We investigated this further by comparing theresults from other electrochemical techniques for the monolayer-modifiedsurfaces (C₁₆SH and C₁₆SH+gD). The capacitance values from theseexperiments are reported in Table IV. The capacitances for C₁₆SH andC₁₆SH+gD SAMs using CV at 0.0 V were 49-53% lower in 0.1 M KNO₃ and46-47% lower in 0.1 M Mg(NO₃)₂ than results obtained at 0.4 V. Forexample, the capacitance of C₁₆SH SAM at 0.0 V in 0.1 M KNO₃ (99.999%)electrolyte was 1.53=0.06 μF/cm², but is 3.05=0.07 μF/cm² at 0.4 V.However, our value for CV at 0.0 V is still higher than Plant, reportsthe capacitance for C₁₆SH monolayer in 0.010 M KCl is about 1.12 μF/cm²using ac impedance at 0.0 V.9 We also performed ac impedance at 0.0 V.The capacitance the C₁₆SH SAMs (0.90=0.08 μF/cm²) is in good agreementwith those previously reported by others using the same technique.

[0167] The results from different electrochemical methods indicate thatcapacitance determinations are very technique-dependent. Because theC₁₆SH SAM-capacitance that we obtained by ac impedance is comparable toliterature values, we conclude that our sample preparation proceduremust be valid. We have only obtained a complete set of capacitance datafor both monolayers and blamers using CV at 0.4 V (Table III). However,SAMs prepared from solutions of C₁₆SH+gD exhibit the same trends incapacitance when compared to pure C₁₆SH SAMs, regardless of thetechnique (see Table IV). Thus, although the absolute values are higherthan those determined by other electrochemical methods, the relativevalues in Table III show the same trends.

EXAMPLE 5

[0168] Chemicals and Materials

[0169] A gold coin (Canadian Maple Leaf, 99.9%) and chromium-platedtungsten rods (R. D. Mathis) served as sources for thermal evaporation.Silicon wafers were obtained from Silicon Quest Internat (Santa Clara,Calif.). Absolute ethanol was obtained from Ultra Scientific.Hexadecanethiol (C₁₆SH), methanol, acetonitrile, chloroform,tetrabutylammonium hexafluorophosphate (TBAPF₆, 96%), silver nitrate,and lead nitrate were obtained from Aldrich Chemical Co. The TBAPF₆ wasrecrystallized from ethanol and dried over night in vacuum at 107° C.(22) Magnesium nitrate hexahydrate, potassium nitrate, potassiumhydroxide, sulfuric acid, lithium perchlorate, and 30% hydrogen peroxidewere obtained from Fisher Scientific. Gramicidin D (gD) was obtainedfrom Sigma Chemical Co. Dimyristoyl phosphatidylcholine (DMPC) wasobtained from Avanti Polar Lipids (Alabaster, Ala.). The10-(ferrocenylcarbonyl)-decanethiol, FcCOC₁₀SH, was synthesized andpurified as described in Everett et al. Purified gA was donated by RogerE. Koeppe, II, and his research group (University of Arkansas,Fayetteville, Ark.). Deionized (DI) water used was purified with aMilli-Q system. Unless otherwise specified, chemicals were used asreceived.

[0170] Fabrication of Working Electrodes

[0171] Silicon wafer substrates were cleaned for approximately 15-20minutes in a solution of 7:3 (v/v) concentrated H₂SO₄ and 30% H₂O₂.respectively, and rinsed thoroughly with DI water. The wafers were driedunder N₂ and oven-dried at 100° C. for 10 min. About 100 Å of chromiumwas thermally evaporated as an adhesion layer followed by 1500-2500 Å ofgold using an Edwards E306A Coating System.

[0172] Derivatizing Solutions and Monolayer Preparation

[0173] Solutions of 1 mM C₁₆SH were prepared by first filtering C₁₆SHthrough a plug of alumina, followed by diluting with 100% ethanol.Solutions of 1 mM FcCOC₁₀SH were prepared in 100% ethanol. Immediatelyafter preparation, solutions were purged for 12-25 minutes with andstored under Ar to minimize disulfide formation. Gramicidin-containingorganothiol solutions (C₁₆SH+gD or FcCOC₁₀SH+gD) were prepared prior touse by dissolving gD or purified gA in the organothiol solution. Unlessotherwise specified, derivatizing solutions consisted of a mole ratio of10 organothiol to 1 gD. Gramicidin D is a mixture of gA. gB and gC. inwhich gA is a major component of the mixture (−85%). Because similarresults were obtained for both gD and purified gA, we do not distinguishbetween gA and gD in this paper.

[0174] SAMs were formed by soaking gold electrodes in derivatizingsolutions for about 24 h under Ar. Electrodes were removed from solutionand rinsed with water or ethanol prior to performing experiments.

[0175] Vesicles and Bilayer Preparation

[0176] Solutions of vesicles of DMPC+gD (mole ratio: 28 DMPC to 1 gD)were prepared. Lipid (140 μmol-50 μmol) was added from stock methanolsolution to dried gD (10 nmol-1 μmol). The total volume was brought to200 μL by the addition of a 50:50 mixture of methanol and chloroform.

[0177] The solution was mixed and the resulting suspension was driedunder vacuum overnight to remove the organic solvent. The driedgramicidin/lipid mixtures were resuspended in 500 μL of Ar-purged waterand sonicated for 2 h at 55° C. using a Branson W-185 cell disrupter(power level 5) fitted with a Mode1431-A cup horn accessory. Sampleswere centrifuged in an Eppendorf Centrifuge 5415C at 12,500 rpm for 5min at room temperature. The gramicidin concentrations in thesupernatant were determined by measuring the absorbance at 280 nm usinga HP 8452A Diode Array spectrophotometer. Solutions of vesicles of DMPCalone (no gD) were prepared as above. The conformation of gD in vesicleswas determined by circular dichroism (CD) measurements that wereobtained at room temperature using a JASCO 710A spectrometer.

[0178] To form hybrid blamers, electrodes were first modified withself-assembly from C₁₆SH and C₁₆SH+gD solutions and rinsed with ethanol.The SAM-coated electrodes were soaked in aqueous suspensions of eitherDMPC or DMPC+gD vesicles for about 24 h and rinsed with water. Hybridblamers were prepared immediately prior to use.

[0179] Our supported hybrid blamers consist of a first layer that is aSAM of hexadecanethiol and a second layer formed from vesicles of DMPCthat are suspended in an aqueous Solution. The advantage of thesestructures is that they better mimic biomembranes than a monolayer. Moreimportantly, the vesicles provide a convenient way to deliver membraneproteins to a surface. Unbound vesicles can be rinsed away with water.Therefore, the proteins which are only slightly soluble in water andwhich are incorporated into the film will not be rinsed away. DMPC waschosen because it is known to form vesicles in which gA is in a channelforming configuration. Each DMPC consists of two butadecyl chains and ahydrophilic head group containing ammonium and phosphate moieties.

[0180] Supported Hybrid Blamers Formed from SAMs and Vesicles

[0181] Blamers were constructed by allowing phospholipid vesicles toassemble from an aqueous suspension onto ethanol-rinsed, SAM-modifiedelectrodes (Graph 22a). CD spectra indicate that gA in the vesicles isin the β channel conformation (Graph 22b). The β channel conformationgives a unique CD spectrum which is characterized by positive peaks at218-220 nm and 235-236 nm, a positive minimum at 229-230 nm and anegative ellipticity below 208 nm (Graph 23). A water rinse rather thanan organic solvent rinse, flushes away free vesicles but will not removegD that has partitioned from vesicles into surface-confined films.

[0182] Film Thickness

[0183] Ellipsometry results for monolayers and blamers are shown in thethickness of films of C₁₆SH/DMPC is 21-25 Å higher than for C₁₆SH alone,essentially twice that of monolayers. The layer of DMPC should have athickness of about 19.6 Å. The experimental C value for DMPC is slightlylarger than the predicted value. This difference might narrow if a moreaccurate refractive index were used. The small deviation from thepredicted value may be due to minute differences between the structureof supported hybrid blamers and planar phospholipid blamers. Thethickness of films of C₁₆SH+gD/DMPC+gD is within error of those withoutgD. The gD must not cause a large enough change in film structure ineither monolayers or blamers to affect thickness.

[0184] These data show more convincingly than the capacitance data thatthe DMPC-formed films are indeed blamers, and not disorganized filmscomposed of multiple layers of organothiols and phospholipids.

[0185] Electrochemical Response to Fe(CN)₆ ³⁻.

[0186] The electrochemical sensitivity to the redox probe Fe(CN)₆ ³⁻ wasinvestigated for monolayers (ethanol-rinsed) and hybrid blamers. Fe(CN)₆^(3−/) ⁴⁻ is a better redox couple than Ag^(+/0) and Pb^(2/0) because itstays soluble in Solution. However, Fe(CN)₆ ^(3−/4−) is much larger andhas a higher charge than the elemental ions. Thus, it is less likely topermeate through small defects in the films. Graph 2a compares thefaradaic response of Fe(CN)₆ ³⁻ at bare and modified electrodes. At baregold, the Fe(CN)₆ ³⁻ gives the typical one-electron, reversibleelectrochemical response. Monolayer and bilayer-modified electrodesblock Fe(CN)₆ ³⁻ from reaching the surface. FIGS. 5b and 5 c showexpanded CV responses for modified electrodes in the absence andpresence of gD, respectively. There is a small faradaic current in thebilayer film that contains gD. This could be due to permeation ofFe(CN)₆ ³⁻ through the film and due to defects caused by the presence ofgD.

[0187] Capacitance in KNO₃ and Mg(NO₃)₂ Electrolyte

[0188] Capacitance values in KNO₃ and Mg(NO₃)₂ were compared to evaluatethe relative permeation of elemental mono- and dications through thefilms. This should be less destructive than electrochemical depositionand oxidation of Ag⁺ and Pb²⁺.

[0189] Capacitance values for clean, bare Au electrodes in 0.1 M KNO₃and 0.1 M Mg(NO₃)₂ are 7.3.8±1.7 and 51.0±2.2 μF/cm², respectively. Thecapacitance for KNO₃ is close to that predicted by theory, 72 μF/cm²,for a 1:1 electrolyte at 0.1 M. The capacitance in Mg(NO₃)₂ should behigher than in KNO₃, because there are three ions for every molecule,instead of two, and one of the ions has a +2, instead of a +1, charge.However, the Mg(NO₃)₂*6H₂O is hygroscopic, and thus, the actualconcentration should be much lower than 0.1 M. This could yield acapacitance that is lower than expected in Mg(NO₃)₂, offsetting thecontributions from increased charge and ion number. We obtainedcomparable capacitance values for Mg(NO₃)₂ and KNO₃ at annealed gold (noadhesion layer) on mica and on gold (with a Cr adhesion layer) on glass.Thus, we are certain that the electrodes and the silicon watersubstrates are not adding unusual capacitive behavior. Reports oftypical C values for bare gold in the literature give values from about30 μF/cm² to 100's μF/cm², which supports the values that are reportedhere. Capacitance is highly dependent on composition of the electrolyte,solvent, nature of the electrode surface, and applied potential. It isimportant to note that our capacitance values and the trends withmodification are consistent and reproducible.

[0190] Graph 1 shows a comparison of the CV response at 0.12 Vs⁻¹ in 5mM Ru(NH₃)₆ ³⁺ and 0.5 MKNO₃ from a (a) 10 um PDM (b) 14 um RDM, and (c)55 um RDM.

[0191] Graph 2 shows a comparison of maximum current taken from CV in 5mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃ at a 55 um RDM to radial and lineardiffusion models; (b) is an enlargement of the region between 0.01 and10 Vs⁻¹.

[0192] Graph 3 shows a comparison of maximum current from CV in 5 mMRu(NH₃)₆ ³⁺ and 0.5 M KNO₃ at a 14 um RDM to radial and linear diffusionmodels; (b) is an enlargement of the region between 0.01 and 10 Vs Ru⁻¹.

[0193] Graph 4 shows the quality of fabrication and seal as determinedby the dependence of capacitance density on scan rate. The chargingcurrent was measured from cyclic voltammograms in 0.5 M KNO Ru₃.Comparison is made to that from Au macroelectrode.

[0194] Graph 5 shows a comparison of chronamperometric responses of aplanar and disk microelectrode in 5 mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃ Thesolution is (a) static, (b) stirred at 70 rpm and (c) stirred at 150rpm.

[0195] Graph 6 shows an overlay of cyclic voltammograms (CVs) collectedat the tubular nanoband electrode (TNE) and RDM inside of a singlecavity.

[0196] Graph 7 shows a scan rate study from 0.01-327 Vs⁻¹ for RDMs in asolution of 5.0 mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃.

[0197] Graph 8 is an expanded view of the region from 0.01-10 Vs⁻¹.

[0198] Graph 9 shows a comparison of i_(max) for the disk microelectrodeto theoretical models for radial and linear diffusion.

[0199] Graph 10 shows a Log-log plot of capacitive density as functionof scan rate for a macroelectrode and recessed disk microelectrode andtubular nanoband electrode. Graph 11 shows CVs from 53 (a, b) and 13 (c,d) urn CES collected in 5.0 mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃solution at 0.1Vs⁻¹. (a) and (c) are from the RMD and (b) and (d) from the TNE. Astandard Ag/AgCl (Sat'd KCL) reference and platinum counter electrodeswere used.

[0200] Graph 11 compares CVs from an RMD and TNE in both 53 (a, b) and13 μm (c, d) cavities.

[0201] Graph 12 shows three CVs (1, 5, and 10th cycles) collected atwith a 53 um CES in 5.0 mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO₃ at 0.5 Vs⁻¹. Thetubular nanoband electrode was modified to serve as a referenceelectrode. The disk was used as the working electrode. Bold larger #5served as the counter-electrode.

[0202] Graph 13 shows CVs from a 13 um CES for 10 and 1 ul samples 5.0mM Ru(NH₃)₆ ³⁺ and 0.5 M KNO ₃of cycled at 0.1 Vs⁻¹. The self-centeredelectrochemistry in the small drops was carried out with electrodesdefined as described in Figure caption 3.

[0203] Graph 14 shows CVs from 53 um CES for 10 and 1 uL samples of 4.0mM hydroquinone in pH 6.60 phosphate (0.05M) buffer cycled at 0.1 Vs⁻¹.The self-contained electrochemistry in the small drops was carried outwith electrodes defined as described in Figure caption 3.

[0204] Graph 15 shows a CA for a 53 um CES in 1.0 mM hydroquinone in0.05 M phosphate buffer (pH 7.0). Static (a) and solutions stirred ateither 70 (b) or 150 (c) rpm are shown. Electrochemistry was carried outwith electrodes as defined in figure caption 3.

[0205] Graph 16 shows a CA for a 13 um CES in 5.7 mM Ru(NH₃)₆ ³⁺ and 0.5M KNO₃ solution. Static (a) and solutions stirred at either 70 (b) or150 (c) rpm are shown. Electrochemistry was carried out with electrodesdefined as in Figure caption 3.

[0206] Graph 17 shows the PM-FTIRRAS spectra of (a) ethanol-rinsed SAMsof C₁₆ SH and (b) C₁₆SH/DMPC bilayer. Transmission IR (c) of dried DMPCin KBr pellet.

[0207] Graph 18 shows PM-FTIRRAS spectra of(a) ethanol-rinsed SAM ofC₁₆SH+gD and (b) C₁₆ SH+gD/DMPC+gD bilayer. Transmission IR (c) of driedgD in a KBr pellet. Graph 19 shows PM-FTIRRAS spectrum of a goldsubstrate modified from a solution of C₁₆ SH+gD and water-rinsed.

[0208] Graph 20 shows XPS spectra of ethanol-rinsed SAMs of (a) C₁₆SH,(b) C₁₆SH+gD, and water-rinsed SAMs of (c) C₁₆SH+gD for O(1s), N(1s),C(1s), and S(2p).

[0209] Graph 21 shows XPS spectra of (a) gD powder and (b) DMPC powderfor selected regions: of O(1s), N(1s), C(1s), and P(2p). (Chargecompensation was used).

[0210] Graph 22 shows CV of Ag⁺ and Pb²⁺ at electrodes modified withhexadecanethiol, with and without gD, and rinsed with ethanol or water.

[0211] Graph 23 shows a CD spectra indicating that gA in the vesicles isin the β channel conformation.

[0212] Whereas, the present invention has been described in relation tothe drawings attached hereto, it should be understood that other andfurther modifications, apart from those shown or suggested herein, maybe made within the spirit and scope of this invention.

What is claimed is:
 1. A 3-dimensional microfabricated device whereinedges of a lipid bi-layer are anchored by alknethiol derivitized inneredges of Au layers in an etched region of insulator and wherein a bottomof the device is lined with an insulator layer.
 2. The 3-dimensionalmicrofabricated device of claim 1 having a hole in said bottom forminimizing osmotic effects.
 3. The 3-dimensional microfabricated deviceof claim 1 having a multiple well array.
 4. A method formicrofabricating recessed disk microelectrodes comprising: growing asilicon dioxide film on a silicon wafer by thermal oxidation;spin-coating said silicon wafer with positive photoresist; exposing saidsilicon wafer to ultra violet light through a photolithographic mask;developing said photoresist and leaving a pattern of parallel lines,which are contact leads and microdisk electrodes; depositing a chromiumfilled adhesion layer on said photo resist by thermal evaporation;sonicating said wafer in acetone to dissolve the photoresist and tocause lift-off of said metal on top of said wafer; drying said wafer;spin-coating said wafer with polyimide; polymerizing said polyimide byexposure to ultraviolet light and curing said polyimide to cross-link apolymer thereby formed; depositing a second chromium layer on top ofsaid polyimide by thermal evaporation; spin coating said wafer withpositive photoresist; patterning said photoresist by exposure toultraviolet light through a second photolithographic mask;simultaneously etching the second gold layer and the second chromiumlayer with aqua regia; stripping a remainder of photoresist withacetone; drying said wafer; leaving a covering of gold and chromiumcovering electrode lines with an area over an end of lines left open forcontact purposes; and forming a hole through said second layer of goldenchromium, and through said polyimide layer to expose said first layer ofgold and chromium.
 5. A microfabricated recessed disk microelectrodeproduced by the process of claim 4.